环子孢子蛋白基因检测法对1例间日疟病例的溯源

对2014年1例初诊为河南省本地感染的间日疟病例进行实验室检测与流行病学溯源调查,以明确其感染来源。收集该病例的流行病学资料,分别采用厚薄血膜吉氏染色法、疟疾快速诊断试纸(RDT)法和巢式PCR法检查患者外周血液,并对其环子孢子蛋白(CSP)基因序列进行分析。流行病学调查显示,该患者曾于2013年5月至缅甸停留约1周,同年6月发病,确诊为间日疟,经青蒿琥酯治疗后,疟原虫转阴,症状消失。CSP基因序列分析显示,该患者前后两次发病时的血样扩增出的CSP序列的中央重复区完全一致,与缅甸分离株(Gen Bank登录号为ABS95455和ABS95456)的序列一致性分别为95.1%和100%,与2个河南分离株HN3和HN7(登录号为KP888996和KP889000)的序列一致性分别为88.8%和67.1%。推测该病例为输入性间日疟复发病例。     

Preparation and in vivo evaluation of anti-plasmodial properties of artemisinin-loaded PCL–PEG–PCL nanoparticles

Abstract

Purpose: Artemisinin (ART) has anti-inflammatory, antimicrobial, antioxidant, anti-amyloid, and anti-malarial effects, but its application is limited due to its low water solubility and poor oral bioavailability. In this study, the bioavailability, water solubility, and anti-plasmodial property of ART were improved by PCL–PEG–PCL tri-block copolymers.

Methods: The structure of the copolymers was characterized by ¹H NMR, FT-IR, DSC, and GPC techniques. ART was encapsulated within micelles by a single-step nano-precipitation method, leading to the formation of ART-loaded PCL–PEG–PCL micelles. The obtained micelles were characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). The in vivo anti-plasmodial activity of ART-loaded micelles was measured against Plasmodium berghei infected Swiss albino mice.

Results: The results showed that the zeta potential of ART-loaded micelles was about ?8.37?mV and the average size was 91.87?nm. ART was encapsulated into PCL–PEG–PCL micelles with a loading capacity of 19.33?±?0.015% and encapsulation efficacy of 87.21?±?3.32%. In vivo anti-plasmodial results against P. berghei showed that multiple injections of ART-loaded micelles could prolong the circulation time and increase the therapeutic efficacy of ART.

Conclusion: These results suggested that PCL–PEG–PCL micelles would be a potential carrier for ART for the treatment of malaria.     

Complement C5‐deficient mice are protected from seizures in experimental cerebral malaria

Studies have demonstrated that the membrane attack complex (MAC) of complement can evoke seizures when injected directly into rodent brain. In the course of studies that examine the role of complement in the development of experimental cerebral malaria (ECM), we observed fewer seizures in mice deficient in C5, a component required for MAC formation. To determine if the MAC contributed to the tonic–clonic seizures characteristic of ECM, we performed long‐term video–electroencephalography (EEG) on C5^?/? mice with Plasmodium berghei ANKA‐induced cerebral malaria and observed significantly reduced spike and seizure frequency compared to wild‐type mice. Our data suggest a role for the MAC in malaria‐induced seizures and that inhibition of the terminal complement pathway may reduce seizures and seizure‐related neurocognitive deficits.     

重组酶介导等温扩增技术检测疟原虫方法的建立和评价

目的 建立一种重组酶介导等温扩增技术（RAA）检测疟原虫的方法。方法 针对疟原虫属保守18S小亚基核糖体RNA （18S rRNA）基因设计特异性引物,筛选确定最佳引物对组合并建立疟原虫重组酶介导核酸等温扩增体系。通过该RAA体系检测疟原虫标准质粒、4种疟原虫[恶性疟原虫( Plasmodium falciparum, P.f )、间日疟原虫( P. vivax, P.v)、三日疟原虫( P. malaria, P.m)、卵形疟原虫( P. ovale, Po)]病原体以及其他6种输血传播寄生虫（婴儿利士曼原虫、杜氏利士曼原虫、刚地弓形虫、溶组织阿米巴虫、犬吉氏巴贝西虫、田鼠巴贝虫）,以评估该体系的灵敏度和特异度。 结果 筛选出一组扩增效果较好的引物对,整个RAA体系使用最佳引物对在37 ℃,20 min即可完成扩增,对标准质粒的检测下限为10^-2 copies/μL,恶性疟原虫、间日疟原虫和卵形疟原虫为10² copies/μL,三日疟原虫为10 copies/μL。RAA检测体系未与其他输血传播寄生虫产生交叉反应,特异性良好。应用RAA方法检测外籍学生献血者标本,结果与荧光定量PCR结果一致。结论 成功建立了一种快速、简便和特异的疟原虫RAA检测方法,将会给血液筛查和一些基层偏远医院的临床检测提供很大帮助。     

The Plasmodium liver-stage parasitophorous vacuole: A front-line of communication between parasite and host

The intracellular development and differentiation of the Plasmodium parasite in the host liver is a prerequisite for the actual onset of malaria disease pathology. Since liver-stage infection is clinically silent and can be completely eliminated by sterilizing immune responses, it is a promising target for urgently needed innovative antimalarial drugs and/or vaccines. Discovered more than 65 years ago, these stages remain poorly understood regarding their molecular repertoire and interaction with their host cells in comparison to the pathogenic erythrocytic stages. The differentiating and replicative intrahepatic parasite resides in a membranous compartment called the parasitophorous vacuole, separating it from the host-cell cytoplasm. Here we outline seminal work that contributed to our present understanding of the fundamental dynamic cellular processes of the intrahepatic malarial parasite with both specific host-cell factors and compartments.     

CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite

Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47^hi)-expressing young RBC. Importantly, C57BL/6 CD47^−/− mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47^−/− mice was associated with a higher percentage of splenic F4/80⁺ cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47^hi young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC.Malaria, caused by Plasmodium parasites, remains a major cause of mortality and morbidity in the developing world. Among the four principal human Plasmodium species, Plasmodium falciparum is the most virulent, being responsible for more than 90% of malaria-associated deaths. Likewise, Plasmodium species that infect rodents and nonhuman primates also differ widely in their fulminant nature and in the mortality they cause (1–3). How different Plasmodium species have evolved to exhibit this wide array of virulence and disease severity remains one of the major unsolved questions in malaria biology and pathogenesis.One important factor that is associated with Plasmodium parasite burden and disease severity is the age constraint of the host red blood cells (RBC) they infect. The age-based preference for restricted invasion of RBC by the Plasmodium parasite is characterized as young RBC (reticulocyte), aged RBC (mature), or both young and aged RBC. Plasmodium species that preferentially infect and grow inside young RBC generally cause a low-grade, self-resolving infection that is rarely fatal (e.g., Plasmodium vivax and Plasmodium ovale), whereas those that infect both young and aged RBC cause more fulminant infection that can be fatal in the absence of immunity (e.g., P. falciparum) (1, 4–6). Thus, along with host genetic background and immune response, restriction for age-specific RBC invasion is a major determinant of the severity and outcome of malaria infection.Malaria parasites have evolved to use redundant receptors and pathways to invade the RBC. For example, sialic acid (7) and Duffy antigen (8) are the major RBC receptors for invasion of P. falciparum and P. vivax, respectively, although other receptors and invasion pathways are known to exist (9, 10). Although a redundancy in RBC receptor use would ensure successful invasion by mitigating the effects of polymorphism and immune targeting, the reasons behind the RBC age-based preference for invasion are not fully clear and remain a subject of debate.Survival of normal cells through the course of their life cycle is essential to maintain homeostasis, and aberrant cells (e.g., senescent or foreign antigen-expressing cells) are eliminated through a sophisticated programmed cell removal system that relies on the recognition of self and nonself determinants (11). CD47, a cell surface molecule in the Ig superfamily, is ubiquitously expressed on many cell types, including RBC, and is a marker of self to avoid early clearance by phagocytic cells through ligation of signal regulatory protein alpha (12). In contrast, altered expression or conformational changes in CD47 may lead to a molecular switch that triggers a phagocytic signal to remove aged or damaged cells (11). Recent studies have shown that the level of CD47 expression is higher in progenitor cells and declines as they undergo maturation and are subsequently aged (13). This age-dependent difference in CD47 expression shields young cells but allows clearance of aging and damaged cells from the system.CD47 is overexpressed in cancer cells (11, 14, 15), and the CD47–signal regulatory protein alpha interaction is considered a major pathway of immune evasion by tumor cells (15). Administration of anti-CD47 antibodies enabled the phagocytosis of tumor cells in vitro, reduced their growth, and prevented the metastasis of human patient tumor cells (14). In this article, using the murine Plasmodium yoelii nonlethal model, we provide quantitative evidence for age of RBC as the basis for the survival and growth of malaria parasites and provide supporting data that suggest that P. yoelii nonlethal parasites prefer to grow inside younger RBC, which allows them to evade immune clearance by phagocytic cells through a CD47-mediated process, and that CD47 modulates the clearance of malaria infection. To our knowledge, this is the first report that provides a molecular basis for the age-dependent preference for infection of RBC by a Plasmodium parasite and sheds light on its implications for the severity of malaria infection in a host.     

Protective immunity to malaria: studies with cloned lines of Plasmodium chabaudi and P. berghei in CBA/Ca mice. I. The effectiveness and inter-and intra-species specificity of immunity induced by infection

CBA/Ca mice were immunized by infection with cloned lines of Plasmodium berghei (isolates ANKA, KSP-11). Plasmodium chabaudi chabaudi (AS, CB) or Plasmodium chabaudi adami (DS) and then challenged with either homologous or heterologous parasites. Protective responses were assessed in immune mice relative to the controls by their ability to (i) extend the time taken for the mean parasitaemia to reach a predetermined level (1% or 0.1%) (ii) reduce peak parasitaemia (iii) resolve the parasitaemia sooner and/or (iv) control or eliminate recrudescences. At both the inter- and intra-species level, immunity appeared largely specific for the cloned line inducing it. At the interspecies level marginally effective cross-immunity was sometimes evident, thus P. berghei KSP-11 immune mice displayed some immunity against P.c. chabaudi AS, although immunity to this parasite was relatively ineffective against P. berghei ANKA or KSP-11. Cross-immunity was more apparent between the subspecies P.c. adami and P.c. chabaudi and between cloned lines of the latter parasite derived from the AS and CB isolates. These data reflect considerable inter- and intra-species structural and immunogenic differences in certain antigens of parasitized erythrocytes and merozoites, which have been identified in a number of murine malarias and associated with protective immunity. Similar differences recently identified in the equivalent antigens of the human parasite P. falciparum may therefore have important implications for protective immunity in man.     

Distinction of recrudescences from new infections by pcr-rflp analysis in a comparative trial of cgp 56 697 and chloroquine in Tanzanian children.

Summary objective To test the efficacy of a new compound drug (CGP 56 697) against acute, uncomplicated falciparum malaria. method Reappearing parasites were analysed by PCR-RFLP within a randomized controlled trial. 130 patients received chloroquine and 130 patients were treated with CGP 56 697. Samples from 96 patients with parasitological failure were tested by PCR-RFLP for MSP2 of Plasmodium falciparum. Seven days after treatment 32 patients of the chloroquine control group with reappearing parasites were tested by PCR and one infection was unequivocally determined as a new infection. After 7 days, in the CGP 56 697 group, 6 samples were tested in which one new infection was identified. Similar observations were made one and three weeks later in both groups. results Although a high multiplicity of infections on admission was observed, there was no significant correlation between multiplicity and either recrudescence or new infection. Patients in both treatment groups with subsequent recrudescent parasites had higher initial mean parasite densities than patients who cleared. Those of the patients with recrudescent parasites who were treated with CGP 56 697 had higher initial parasite densities than those treated with chloroquine. The rate of re-infection increased with time as expected in holoendemic areas and appeared to be higher in chloroquine patients. Generally, CGP 56 697 showed a superior clearance rate, successfully cleared higher parasite densities and suppressed new infections over a longer period of time. conclusion The PCR analysis confirmed that reinfections beyond day 7 are significant in areas highly endemic for malaria and showed the necessity of excluding these when estimating 14 day clearance rates. Provided new infections are excluded, the 28-day clearance rate can also be used to determine the efficacy of antimalarial drugs in highly endemic areas, and adds to our knowledge of drug resistance and dynamics of infections in people living in such areas.     

Dipstick urinalysis findings in children with Plasmodium falciparum in the South Tongu District: A case-control study

Background:

Malaria ranks among the major health and developmental challenges facing some of the poorest countries in tropical and sub-tropical regions across the globe. We determined urinary abnormalities and its relationship with parasite density in children ≤12 years with Plasmodium falciparum infection.

Materials and Methods:

From December 2013 to March 2014, we randomly recruited 116 participants comprising 58 malaria patients (cases) and 58 healthy controls from the Comboni Mission and the Sogakope District Hospitals both in the South Tongu district. Blood was collected for the estimation of hemoglobin and total white blood cells; thick and thin blood films were used for the determination of malaria parasite density. Urine was collected for the measurement of the various biochemical components using the automated urine analyzer. A pretested questionnaire was used to obtain demographic and clinical data.

Results:

Urine protein (P < 0.001), blood (P < 0.001), bilirubin (P < 0.001), urobilinogen (P < 0.001), and ketones (P = 0.001) were significantly higher in individuals with P. falciparum infection than in healthy controls. Proteinuria (P = 0.247; r = 0.155), hematuria (P = 0.142; r = 0.195), bilirubinuria (P = 0.001; r = 0.438), urobilinogenuria (P = 0.876; r = 0.021), and ketonuria (P = 0.136; r = 0.198) were positively correlated with malaria parasite density; however, only bilirubinuria was significantly higher at higher parasitemia.

Conclusion:

Malaria has a significant effect on the chemical composition of urine with bilirubin positively correlated with parasite density. Dipstick urinalysis can be used together with light microscopy in resource-limited malaria-endemic areas to accurately diagnose falciparum malaria infection.     

Transdermal Diagnosis of Malaria Using Vapor Nanobubbles

A fast, precise, noninvasive, high-throughput, and simple approach for detecting malaria in humans and mosquitoes is not possible with current techniques that depend on blood sampling, reagents, facilities, tedious procedures, and trained personnel. We designed a device for rapid (20-second) noninvasive diagnosis of Plasmodium falciparum infection in a malaria patient without drawing blood or using any reagent. This method uses transdermal optical excitation and acoustic detection of vapor nanobubbles around intraparasite hemozoin. The same device also identified individual malaria parasite–infected Anopheles mosquitoes in a few seconds and can be realized as a low-cost universal tool for clinical and field diagnoses.