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Entamoeba histolytica trophozoites isolated in Robinson's medium from faeces containing cysts, transported from India, have been tested in vitro against the drugs furazolidone, iodochlorhydroxyquin, and both drugs combined as Dependal. Results show the levels at which drug action is significant and that interaction between the drugs occurs at two concentrations.
A country-wide stool survey of The Gambia showed the prevalence of Entamoeba histolytica cysts to range from 13.7% up-country in the dry season to 52.3% near the coast. A longitudinal survey showed a near 100% infection rate over one year and a sharp rise in prevalence as the rains commence with an equally sharp fall as the rains progress. Specific antibody levels are elevated and reasonably constant through the year. Carriers generally show no specific lymphocyte reactivity to amoebic antigen but consistently parasite-negative individuals tended to show elevated lymphocyte reactivity. Attempts to discover the presence of cysts in the environment of villages by cultivation of specimens of water, soil, food, flies and washing from clothes and hands generally failed though E. histolytica was recovered once from a well.
Cultures of 85 stocks of Entamoeba histolytica were compared by electrophoretic patterns of three enzymes: glucosephosphate isomerase, phosphoglucomutase and L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating). Four groups of E. histolytica stocks have been distinguished. 18 of these stocks were derived from individual patients with clinical amoebiasis and all of these were of one enzyme group, which did not occur among the other stocks.
Amoebic antigen prepared from axenic Entamoeba histolytica (NIH:200 strain) grown at 37 degrees C in TPS I medium was chromatographed on a Sephadex G-200 column. Three fractions (F-I, F-II, F-II) were obtained, the molecular weights of which ranged from 650,000 to 1,450. Fractions were concentrated by lyophilization and antisera against each fraction and whole antigen were raised in rabbits. Partially purified fractions were used in various serological tests and their reactivities were compared with whole amoebic antigen. Main haemagglutinating and precipitating activities were confined to Fraction I, which had 14% of total protein and the highest molecular weight (650,000). 5 micrograms/ml of protein contained in this fraction gave antibody titres comparable to those obtained with whole antigen with 40 micrograms/ml or protein. Other fractions, although they had precipitin activity, lacked haemagglutinating activity. The protective efficacies of these fractions are being investigated in our laboratory.
Axenic amoebic antigen was chromatographed on Sephadex G-200. Two peaks divided into three fractions were obtained which were concentrated by lyophilization. Guinea-pigs were given a series of subcutaneous injections of crude Entamoeba histolytica antigen and its chromatographic fractions mixed with Freund's complete adjuvant. After three immunizing doses, the animals were challenged with intracaecal injection of a virulent isolate of amoebae (Strain No. H-25) and were killed seven days later. The antibody response was studied by indirect haemagglutination (IHA) and counter-current immunoelectrophoresis (CIEP) before intracaecal inoculation and at the time of killing. Fraction I, which had the highest molecular weight, stimulated high levels of antibodies in guinea-pigs and showed 92% protection. Only one animal of 12 inoculated had gross lesions and these were very mild. The caecal contents of three of the 11 animals without gross lesions were positive for amoebae but no tissue invasion was observed. Partial protection was observed with other antigens--30% with whole amoebae, 18% with Fraction II and 30% with Fraction III. In most there were multiple ulcers of the deep penetrating type and amoebae either penetrating and invading the muscular layer or the lytic process extending up to the muscular layer. In one animal, immunized with whole amoebic antigen, multiple hepatic lesions were also observed and trophozoites of E. histolytica were demonstrated histologically.
Stocks of intestinal amoebae isolated from hospital patients in Mexico City and grown in monoxenic culture were compared among themselves and with those already described (SARGEAUNT & WILLIAMS, 1979), using the electrophoretic patterns of four enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM), L-malate:NADP+ oxido-reductase (oxalacetate-decarboxylating) (ME) and hexokinase (HK). New isoenzyme groups (SARGEAUNT & WILLIAMS, 1979) of all the amoebae, including Entamoeba histolytica have been demonstrated. Amongst these have been found seven more groups of E. histolytica, two new groups of E. hartmanni, one new group of Dientamoeba fragilis and one new group of E. coli. Of the seven new groups of E. histolytica three are known to originate from patients with clinical amoebiasis whilst the remainder are from asymptomatic subjects. Only 11.2% of the 125 isolations were associated with clinical amoebiasis, and these are clearly distinguished from the isolations from asymptomatic patients by their electrophoretic isoenzyme pattern.
The virulence of 70 isolates of Entamoeba histolytica was studied in the Wistar strain of albino rat by intracaecal inoculation of amoebae. The results were evaluated by the Neal scoring system, histopathological grading of ulcers and virulence indices. It was found that isolates from acute amoebic cases could infect and produce ulcers in significantly more animals than could the isolates from asymptomatic cyst passers. The virulence indices of isolates from acute cases were also significantly higher as compared to those from asymptomatic cyst passers. However, there was no significant difference between virulence indices of isolates from acute cases and non-dysenteric amoebic colitis. This study also showed that Neal scores failed to correlate with the type of pathology produced by the amoebae in the caecum. It is felt that virulence indices which depend upon the histopathological lesions should also be taken into consideration. The serological response of the patients tended to correlate with the virulence indices of isolates but only minimally. The results thus contradict a common belief amongst clinicians that higher levels of anti-amoebic antibodies reflect severe disease, as compared to low levels of antiamoebic antibodies.
A method for detecting Entamoeba histolytica trophozoite antigens in aqueous solution is described. This involves coating plastic microtitre plates with a 'catching antibody', specific rabbit anti-E. histolytica antibody (SRAE). After adding the test material the presence of antigen is determined using two additional heterologous antibody layers, one of 'developing antibody', in this case human anti-E. histolytica immunoglobulin (HAE), which is followed by a final layer of peroxidase conjugated sheep anti-human immunoglobulin antibody (SH-HRP). The specificity and sensitivity of the assay was investigated both in the model system and using stool samples from infected patients. In the model system, the test had a sensitivity equivalent to detection of approximately one amoeba per microscope coverslip (18 mm X 18 mm). Little cross reaction was observed with other intestinal parasites common to the area of Bangladesh from which the stool samples were taken. The possible use of this method in large scale screening of stool samples and in the detection of circulating antigens is discussed.
A comparison was made of susceptibility to lysis by human sera among five non-pathogenic and 11 pathogenic strains of Entamoeba histolytica already characterized into zymodemes. The nonpathogenic strains were found to be uniformly susceptible to lysis. Nine of 11 pathogenic strains, including five strains isolated from liver abscesses, were found to be resistant to lysis by serum under identical conditions. Resistance to complement-mediated lysis may be an inherent property of most pathogenic strains and may prove to be a necessary virulence factor for dissemination.
An infectivity titration technique is described for the measurement of the viability of Entamoeba histolytica. Using this method an optimal cryopreservation technique has been developed. When amoebae from young cultures were equilibrated with 7.5% dimethylsulphoxide for 15 minutes at 37 degrees C, cooled to -100 degrees C at 1 degree C per minute, and stored in liquid nitrogen, 1 to 2% regularly survived after thawing rapidly at 37 degrees C and, on occasions, 10 to 12% survived.
The antifungal antibiotic sinefungin, a potent inhibitor of methyl transferases and a potential inhibitor of polyamine biosynthesis, exhibited marked cytolytic activity against Entamoeba histolytica in vitro with respect to all the amoeba strains tested. Strains of high and low virulence displayed equal sensitivity to the antibiotic. Minimal Amoebicidal Concentration was 5 to 10 micrograms/ml.
Sera from 15 patients with Entamoeba histolytica infection were tested for anti T-cell antibodies by assessing cross-reacting specificities with the antigens defined by an anti-Ia hybridoma antibody. T cells prepared by sheep erythrocyte rosetting were preincubated with the test sera and then with the anti-Ia antibody. Binding of the specific monoclonal antibody was assessed by rosetting with ox erythrocytes conjugated with goat anti-mouse IgG. Eight sera from amoebic patients were found to block the binding of monoclonal mouse hybridoma anti Ia-antibody to T cells. Blocking of anti-Ia binding was not due to Fc IgG receptor binding by immune complexes nor was it HLA-DR restricted. T cells pre-treatment with the amoebic sera positive for anti-Ia activity showed reduced activity when tested in the autologous mixed lymphocyte reaction (AMLR). The results of our study seem to suggest the existence of specific anomalies of immunoregulation during E. histolytica infection which may play a role in inducing immune disregulation in vivo.
Stocks of Entamoeba histolytica isolated and maintained in a variety of culture media, both axenic and polyxenic, were compared with each other and with strains previously characterized. The present stocks were isolated from subjects living in various cities in India. Using the enzyme patterns of: E C 5319 glucose phosphate isomerase (GPI); E C 11140 L-malate: NADP+ oxidoreductase (oxaloacetate decarboxylating) (ME): E C 2751 phosphoglucomutase (PGM); and E C 2711 hexakinase (HK), developed after electrophoresis, three zymodemes were identified in 54 individual isolations of E. histolytica. Most importantly only one zymodeme associated with pathogenicity occurred, identified 28 times, among the collection, one of which was from a liver abscess.
A promising new ELISA method developed by Root et al. to diagnose amoebiasis by the detection of Entamoeba histolytica coproantigen was tested in Mexico City and was reported to be more than twice as sensitive as microscopy. In this study the same procedure was compared to microscopy for paired specimens from 107 patients in San Francisco. Microscopy was more than twice as sensitive as ELISA: of 12 specimens positive by microscopy only 5 were also positive by ELISA. The ELISA did not detect a single specimen not detected by microscopy. Over-all, there was a 93% agreement between the two tests: 5% were positive and 89% negative by both tests. Possible reasons for the differences in the San Francisco and Mexican City findings are discussed.
Groups of athymic rnu rats were found to be susceptible to SAW 408 Entamoeba histolytica introduced intra-intestinally. The same strain of rat could be infected orally with cultured trophozoites using a gastric secretion inhibitor, cimetidine, as a pretreatment. Groups of Lac:P and Wistar rats similarly treated were also found to be susceptible although the parasite population growth was slow. A similar regimen attempted on susceptible mice was unsuccessful.
Lethal recognition between Entamoeba histolytica and the tissues and defence systems of the host results in a continuous interplay that determines the development of pathological lesions: (i) we have identified several of the steps and mediators utilized by the trophozoites to destroy host cells by contact-mediated cytolysis; (ii) we have established that the alternative complement system represents the main defence available to the host against the invading parasite. The amoebae recognize target cells by means of a lectin specific for N-acetylgalactosamine-containing surface glycoproteins. This recognition appears to activate the amoeba to release, in the area of contact, an attack complex that induces the host cells to undergo cytolysis. The main component of the attack complex is thought to be amoebapore, an ion-channel forming protein that incorporates spontaneously into target cells leading to their depolarization by creating a pathway for ions to flow down their concentration gradient. The known properties of amoebapore are described. The acquisition of complement resistance by the invading trophozoites is essential for their survival within the host and therefore underlies virulence. The resistance to complement killing is not a permanent property of the amoebae. It is lost during axenization and reappears on passage through the host or when the trophozoites are grown axenically in the presence of active complement.
Twelve antimicrobial agents were tested for use in axenic cultures of Entamoeba histolytica and Giardia lamblia. Ten did not inhibit parasite growth at concentrations of at least two-fold above the effective antibacterial or antifungal levels. We have found the combination of piperacillin (500 micrograms/ml) plus amikacin (125 micrograms/ml) to be useful in axenization of a new strain of E. histolytica, eradication of multiply drug-resistant contaminants in cultures and prevention of bacterial growth in experiments with non-sterile components.
Entamoeba histolytica was first seen by Lösch (1875), and subsequently described by Schaudinn (1903). Since that time this organism has only been known to divide by binary fision. Observations at molecular level now reveal possible genetic exchange in this species of amoeba.