Entamoeba histolytica cytotoxin: purification, characterization, strain virulence, and protease activity

A heat-labile cytotoxin was isolated from virulent strains of axenically cultivated Entamoeba histolytica. Strains of E. histolytica representing a spectrum of virulence as determined in animal and in vitro models of disease were examined for cytotoxic activity. Extracts of virulent strain HM1 possessed marked cytotoxic activity, those of moderately virulent strain 200 showed intermediate activity, and those of avirulent strains 303 and Rahman showed no activity. The cytotoxin was partially purified from the cell-free supernatant of sonicated E. histolytica HM1 trophozoites by ammonium sulfate precipitation and gel filtration. Cytotoxic activity was stable in a narrow pH range (6-7.2) and in 1 M NaCl, urea, and guanidine. Specific immune rabbit and human antiserum as well as the protease inhibitors aprotinin, pepstatin, and leupeptin inhibited cytotoxicity. The partially purified cytotoxin did not have any detectable degradative enzymatic activities. Thus, virulent strains of E. histolytica possess an immunogenic cytotoxic protein which may be important in the pathophysiology of amoebiasis.     

FTA-巢式PCR方法鉴定溶组织内阿米巴原虫的初步研究

目的建立一种简便,快速的FTA-巢式PCR方法用于鉴定粪便中溶组织内阿米巴原虫(Entamoeba histolytica, E.h).方法 收集门诊腹泻病人新鲜粪便,用光学显微镜进行初步检查.用FTA卡抽提镜检结果为阳性的粪便DNA,根据溶组织内阿米巴原虫的SSU -rRNA序列设计引物,进行巢式PCR扩增,对PCR产物进行琼脂糖凝胶分析,并对阳性产物进行测序和序列比对分析.结果 根据光学显微镜检测结果,挑选了44例镜检结果为阿米巴原虫阳性的腹泻病人粪便.经FTA-巢式PCR扩增,其中20例样本可扩增出427 bp左右的目的条带,目的条带的测序和序列分析结果表明为溶组织内阿米巴原虫.镜检方法与巢式PCR方法的阳性符合率为45.45%(20/44),将E.h与形态学相似的其他内阿米巴原虫进行了鉴定和区分.结论 本研究建立的FTA-巢式PCR方法具有简单,快速,准确等优点,为临床检验和流行病学调查中E.h的鉴别诊断提供了新的技术方法.     

The rhabdoviruses of Entamoeba histolytica and Entamoeba invadens.

Rhabdoviruses have been described in plants, arthropods and vertebrates including man. Members of the group are of agricultural, veterinary and medical importance. The presence of a rhabdovirus in Entamoeba histolytica and Entamoeba invadens is the first record of their existence within protozoa. The morphology of this virus is described and its significance discussed, in relation to a possible lysogenic state and pathogenecity of Entamoeba species.     

Destruction of normal human eosinophils by Entamoeba histolytica

We evaluated the in-vitro interaction of normal human eosinophil leucocytes and a virulent strain of Entamoeba histolytica (HM1-IMSS) in the presence of immune serum. At a 10:1 (eosinophil:amoeba) ratio a significant time-dependent destruction of eosinophils was found from the first hour onward, and a similar, albeit weaker, cytopathic effect was found in the 200:1 ratio mixtures, with some delay in the microscopic evidence of such effect. Results were unaffected by serum factors, and amoebae emerged virtually unharmed throughout these experiments, again regardless of the presence of serum factors. These results indicate that, as with neutrophil leucocytes, virulent E. histolytica is capable of destroying normal human eosinophils in vitro.     

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhood malnutrition. Traditional microscopy approaches are neither sensitive nor specific for Entamoeba histolytica. Antigen assays are more specific, but many cases are missed unless tested by molecular methods. Although polymerase chain reaction (PCR) is effective, the need for sophisticated, expensive equipment, infrastructure, and trained personnel limits its usefulness, especially in the resource-limited, endemic areas. Here, we report development of a recombinase polymerase amplification (RPA) method to detect E. histolytica specifically. Using visual detection by lateral flow (LF), the test was highly sensitive and specific and could be performed without additional equipment. The availability of this inexpensive, sensitive, and field-applicable diagnostic test could facilitate rapid diagnosis and treatment of amebiasis in endemic regions.     

Complement activation by antigenic fractions of Entamoeba histolytica

Complement (C) activation induced by Entamoeba histolytica in normal non-immune human serum was studied by testing in vitro the ability of different fractions of the trophozoites to cause C3 breakdown. Whole trophozoites were found to activate both alternative and classical C pathways. The antibody-independent classical pathway (MgEGTA inhibitable) C activating capacity was found to greatly increase after disruption of the cell membrane by sonication. Subsequent analysis after differential centrifugation and ion exchange chromatography of the membrane/particulate fractions showed, that this activity was not due to DNA, which has been shown to have the similar characteristics, but to other, as yet unidentified components. A rather homogenous membrane fraction obtained by elution with 0.4 M Tris-HCl, pH 8.1 (described as 4M) and some cytoplasmic constituents obtained after gel chromatography retained a moderate degree of alternative pathway C3 activating capacity seen with intact trophozoites. Thus, it seems, that serum contact to the outer surface of E. histolytica trophozoites leads to C activation via both pathways with cell death as the result and to subsequent release of more efficiently classical pathway activating components. These phenomena probably have an important role in the inflammatory process in invasive amoebiasis.     

Susceptibility testing of Entamoeba histolytica

The growth of Entamoeba histolytica in microtiter plates in vitro in a variety of environments with reduced oxygen tensions is reported. With 3% O2, 3% CO2, and 94% N2, the parasite growth in microtiter plates was identical to that in screw-capped culture tubes, as measured by [3H]thymidine incorporation and by quantitative parasite counts. There were no significant differences between the drug concentrations necessary to inhibit parasite growth by 50% based on [3H]thymidine incorporation vs those defined by quantitative parasite counts for the 15 antimicrobial agents tested (including seven drugs used for the treatment of amebiasis). This technique provides a reproducible method to quantitate the activity of potential antiamebic agents in vitro. The isotopic method should be of particular value in defining the metabolism of the parasite and effects of antimicrobial agents on it, whereas the morphologic method may be more valuable for workers with limited resources available to them.     

Usefulness of multiplex-PCR for identification of Entamoeba histolytica and Entamoeba dispar

We evaluated the usefulness of a multiplex-PCR method for differentiation of Entamoeba histolytica and Entamoeba dispar, which are morphologically indistinguishable species. Cultured trophozoites of E. histolytica HM-1: IMSS and E. dispar SAW were used as the positive control. Seven human fecal samples, from which E. histolytica-like cysts were detected by microscopic examination, and three intestinal protozoan parasites, Cryptosporidium parvum HNJ-1, Giardia intestinalis Portland-1, and Blastocytis hominis Nand II, were used for the evaluation of sensitivity and specificity of the PCR method. The other PCR method, which has been used for the diagnosis of amebic infections in Japan, was also performed by using the same samples for the evaluation. In comparison with the conventional PCR method, the multiplex-PCR showed 1) higher sensitivity, 2) the size of diagnostic fragments of PCR products was clearly different in both Entamoeba species, 3) it was possible to perform PCR using a single tube per sample, and then to save the amount of DNA polymerase, 4) no diagnostic amplification products were found in other intestinal protozoan parasites, and 5) E. histolytica specific fragment was amplified in all clinical samples examined. In conclusion, it is considered that the multiplex-PCR method is a useful tool for detection of both Entamoeba species DNA from fecal samples and for the distinction between E. histolytica and E. dispar.     

Growth inhibition of axenic Entamoeba histolytica by tubercidin and ara-A

Several analogs of nucleic acid components including 7-deazaadenosine (tubercidin), adenine 9-beta-D-arabinofuranoside (ara-A), 8-azaguanine, 6-azathymine, 6-mercaptopurine, 6-mercaptopurine arabinoside, 6-mercaptopurine riboside and 1,3-dideazapurine (benzimidazole) were tested for inhibition of growth of an axenic strain of Entamoeba histolytica in 72 hour experiments. Metronidazole and emetine were included in the experiments for comparison. Tubercidin and ara-A, analogs of adenosine, were the most potent of the tested growth inhibitors with amebistatic concentrations of about 0.6 and 3.0 microM, respectively. The other analogs did not significantly inhibit amebal growth below 100 microM. In the present study metronidazole and emetine had amebistatic concentrations, respectively, 17- and 60-fold higher than tubercidin.     

Migration of Entamoeba histolytica under agarose

A procedure for visualizing and quantifying motility of Entamoeba histolytica by migration under agarose is described. Agarose suspended in tissue culture medium 199 supplemented with bovine albumin was poured into plastic dishes and allowed to harden. Six pairs of wells were cut out in a circular configuration. To the inner wells a suspension of E. histolytica in Eagle's Medium (24 X 10(6) cells/ml) was added, and to the outer wells a chemoattractant or the control medium. After overnight incubation at 37 degrees C, the amebae were fixed and stained. The chemotactic and spontaneous migrations were measured in an enlarging projector. Escherichia coli filtrates, suspensions of intact and lysed erythrocytes, and the complement factor C5a acted as good chemoattractants. Both the random and chemotactic motility were correlated to the time of the incubation. Cytochalasin B effected a dose-related inhibition of both chemotactic and random migration, while colchicine caused a decrease of the chemotaxis only. The reproducibility of the method, measured by 10 intra-assay tests, was good. Thus, the described method can be useful for comparative determinations of the motility of different ameba populations. Furthermore, different factors affecting the motility of amebae can be studied.     

Interaction of rheumatoid factor and Entamoeba histolytica

The amoebae's cytotoxicity test and the amoebae's lysis test were used to show possible interactions between rheumatoid factor (RF) and Entamoeba histolytica. Amoebae's cytotoxic activity (ACA) was inhibited by affinity chromatography purified antiamoebae rabbit IgG (RIgG). Enhanced inhibition could be demonstrated with RIgG plus RF. But the same marked inhibition of ACA could be seen when replacing RF by heat inactivated normal human serum as a control. About 50% amoebae's lysis occurred when amoebae were brought together with native normal human serum (NNHS) as a source of complement. Amoebae's lysis increased to 60% when incubated with NHS plus human antiamoebae antibodies. No further augmentation could be obtained by the addition of RF. Using RIgG instead of human antibodies the lysis rate did not increase. Incubation of amoebae, NNHS, RIgG and RF even reduced amoebae's lysis. RF neither has an effect on ACA nor on complement mediated AL in vitro.     

Effect of anabolic steroids on plasma antithrombin III. alpha2 macroglobulin and alpha1 antitrypsin levels.

The effect of seven different anabolic steroids (Ethyloestrenol, Methenolone acetate, Norethandrolone, Methylandrostenediol, Oxymetholone, Methandienone, and Stanozolol) on three alpha-globulin antiprotease inhibitors of thrombin and plasmin was studied in men with ischaemic heart disease. In distinct contrast to the oral contraceptives, five of the six 17-alpha-alkylated anabolic steroids studied produced increased plasma Antithrombin III levels and five produced decreased levels of plasma alpha2-macroglobulin. The effect on plasma alpha1-antitrypsin levels was less clear-cut but three of the steroids examined produced significantly elevated levels. The increased plasma fibrinolytic activity which the 17-alpha-alkylated anabolic steroids induce is therefore unlikely to be secondary to disseminated intravascular coagulation.     

Sensitivity of Entamoeba histolytica and Entamoeba dispar patient isolates to human complement

Twenty-one Entamoeba histolytica and 56 Entamoeba dispar patient isolates were investigated for their sensitivity to the classical and alternative pathway of human complement. E. histolytica and E. dispar patient isolates were differentiated by polymerase chain reaction and hexokinase isoenzyme typing. It was found that 90.3% (±12.0%) of the trophozoites of E. histolytica were lysed after 30 min by the alternative pathway of complement in the presence of 50% human serum (19 isolates showed lysis rates higher than 80%), whereas E. dispar cells were less susceptible to the alternative pathway as 68.8% (±28.2%) of lysis occurred. However, 23 of the E. dispar isolates were lysed between 100 and 80% (90.9%±9.1%), demonstrating that about half of the tested E. dispar isolates were highly sensitive to complement lysis. Only 11 of the E. dispar isolates were proven to be 'resistant' to the alternative pathway of complement and were lysed less than 40%. These results are in conflict to earlier publications, describing resistance of E. dispar to complement lysis (Hamelmann et al. 1992, 1993).     

The destruction of virulent Entamoeba histolytica by activated human eosinophils

Unlike normal (i.e., non-activated) human eosinophils that are unable to destroy virulent Entamoeba histolytica even in the presence of antibodies and complement, activated eosinophils effectively destroy the parasite in vitro without the help of opsonins, yet increase this capacity with their assistance. Many activated eosinophils succumb in the process as well, probably victims of toxic products released by dying amoebae. Human activated eosinophils thus behave more like activated macrophages than like neutrophil polymorphonuclear leucocytes that are notoriously incompetent in dealing with virulent amoebae. As a regular constituent of early inflammatory reactions, and notwithstanding the absence of blood and tissue eosinophilia in invasive amoebiasis, the activated eosinophil may play a role in the defence against E. histolytica.