Entamoeba histolytica and Entamoeba dispar are distinct species; clinical, epidemiological and serological evidence.

The name of the causative organism of invasive amoebiasis, Entamoeba histolytica, was first introduced in 1903, even though this intestinal amoeba had been recognised since 1875. The marked disparity between the number of infected individuals and those with invasive amoebiasis resulted in a number of explanatory hypotheses being proposed. Although none of these were universally accepted, Brumpt's concept of two morphologically identical species gained increasing acceptance 50-60 years later when technology became available to investigate this anomaly. Sargeaunt spear-headed this drive by establishing the value of isoenzyme electrophoresis for studying the host-parasite relationship. From this foundation, incorporation of clinical, epidemiological and serological parameters to studies of the parasite resulted in the conclusion that a species complex comprising two morphologically identical amoebae was implicated with the disease. The two organisms have been named E. histolytica and Entamoeba dispar. The former is a pathogen and is responsible for invasive amoebiasis, while the latter is a gut commensal. Demonstration of the existence of this species complex has subsequently been confirmed by studies on the nucleic acids from several independent laboratories. The acceptance of E. histolytica and E. dispar as distinct species has had a major impact on our understanding of amoebiasis and its clinical management.

Longitudinal study of the antibody response to recombinant Entamoeba histolytica antigens in patients with amebic liver abscess.

Serology is a critical component in the diagnosis of amebic liver abscess. However, in areas endemic for amebiasis there is a high background level of seropositivity for amebiasis (owing to previous infection with Entamoeba histolytica), which may complicate the interpretation of a positive serologic test result. Recently, we reported that serologic tests based on recombinant E. histolytica antigens might offer improved diagnosis of current invasive amebiasis because they apparently differentiated active infection from past exposure to the parasite. To confirm this finding, we have performed a longitudinal study on 20 patients with amebic liver abscess by examining their seroreactivity over time with recombinant versions of two major E. histolytica proteins, the serine rich E. histolytica protein (SREHP), and the 170-kD subunit of the galactose-specific adhesin. We found that more than 50% of the patients examined had become seronegative by one or both recombinant tests within 180 days of their diagnosis of amebic liver abscess. In the case of the recombinant SREHP-based tests, 12 patients had become seronegative 90 days after presentation. In contrast, all patients remained seropositive by a standard conventional test, an indirect hemagglutination test, at more than six months after presentation. Our study shows that patients lose seroreactivity with the recombinant SREHP or 170-kD antigen-based tests more rapidly than with a conventional serologic test; this may make them useful for the serologic diagnosis of amebiasis in endemic areas.

Protection against amebic liver abscess formation in the severe combined immunodeficient mouse by human anti-amebic antibodies.

We have used serum from patients with amebic liver abscess to investigate the role of antibody in the prevention of invasive amebiasis using the severe combined immunodeficient (SCID) mouse model of Entamoeba histolytica infection. The SCID mice were passively immunized with serum or purified antibody from patients with amebic liver abscess 24 hr prior to the direct intrahepatic challenge with 10(6) virulent E. histolytica trophozoites. This treatment reduced the mean abscess size in these animals from 24.5% to 3.5% (P < 0.0001). These results demonstrate that human anti-amebic antibodies are capable of exerting a protective effect in an animal model of amebic liver abscess formation.

Biological evidence of genetic exchange in Entamoeba histolytica.

The demonstration of a new zymodeme of Entamoeba histolytica produced in culture from cloned isolates suggested possible genetic exchange in this parasite. We have attempted to substantiate that finding by using rats as biological hosts. Clones were made from 3 separate isolates of E. histolytica, each established in culture from liver pus or faeces. After enzyme characterization these clones, of zymodemes II, XIV and XIX, were paired in each of the 3 possible combinations and the mixtures injected into the caecum of rats. Clones of new or hybrid zymodemes were produced as well as the original parents, with one exception, the mixture of XIV and XIX, from which only one of the parents was recovered. The hybrids produced included zymodeme XX, observed previously, and zymodeme XI, a naturally occurring zymodeme that has in the past been recovered only from subjects with invasive amoebiasis.

Application of the leucocyte adherence inhibition test to the assessment of cell mediated immunity in invasive amoebiasis.

The leucocyte adherence inhibition (LAI) test was assessed as a means for measuring specific cell mediated immunity (CMI) in invasive amoebiasis. The LAI test and the amoebic gel diffusion (AGD) test were performed on 20 patients admitted to hospital with amoebic liver abscess (ALA) and 24 control subjects with no demonstrable clinical amoebiasis. The AGD results were acceptable for such a study in an endemic area as all the proven ALA subjects gave positive responses while all except one of the controls gave a negative reaction. A percentage adherence in the LAI test of more than 80% was considered to be negative and the mean percentage adherence of leucocytes for the control subjects was found to be 86.0 +/- 4.9 while that for the experimental group was found to be 76.1 +/- 15.4; the difference between these groups is statistically significant. It was noticed that the LAI test response was possibly linked with severity of disease in the ALA subjects. The LAI test has been found to be a relatively rapid test of specific CMI and although fastidious, it does not require the use of radio-isotopes.

Serological differentiation between past and present infection in hepatic amoebiasis.

The indirect fluorescent antibody (IFA) test and the amoebic gel diffusion (AGD) test were assessed as tools for differentiating between past and present infection in invasive amoebiasis; in the case of the IFA test the Entamoeba histolytica-specific IgG and IgM were monitored. In a pilot study in which sera from 56 patients with suspected invasive amoebiasis were tested the IgM was positive in 40% of confirmed intestinal and 83% of confirmed hepatic cases, the IgG and AGD were positive in all confirmed cases. A subsequent study was then carried out, in which the tests were used to monitor longitudinally antibody levels of patients with confirmed amoebic liver abscess, from the time of admission to hospital, to one year after successful treatment. The specific IgM levels became negative sooner than the IgG or AGD, with more than half the subjects giving negative results at six months and all cases becoming negative 46 weeks after treatment. The results of this study suggest that the presence of specific IgM, together with specific IgG and a strongly positive AGD test, is indicative of an active infection; conversely, when the IgM is negative, while the other tests are positive, active disease was usually absent.

A zymodeme study of Entamoeba histolytica in a group of South African schoolchildren.

Using a biphasic culture medium, stocks of intestinal amoebae were isolated from a group of children attending school in Durban, South Africa. These were compared with stocks collected in other areas of the world already characterized using the electrophoretic patterns of four enzymes: glucose phosphate isomerase (GPI), phosphoglucomutase (PGM) L-malate: NADP+ oxido-reductase (oxalacetate-decarboxylating) (ME) and hexokinase (HK). 33% of 94 samples grew Entamoeba histolytica, only one of which gave a pattern indicative of a pathogenic stock. Entamoeba hartmanni, Dientamoeba fragilis and Entamoeba coli were also grown from some samples, increasing the total positive samples for all species isolated to 40%.

Cell-mediated immunity in hepatic amoebiasis.

Cell-mediated immunity (CMI) was measured by blastic transformation of peripheral blood lymphocytes in 26 patients with amoebic liver abscess (ALA) and matched control subjects with no demonstrable clinical amoebiasis. During active disease, the mean mitogenic response, measured by the stimulation index of the patients' lymphocytes to Entamoeba histolytica antigen, was increased (mean +/- SD: 25.98 +/- 46.62 compared with 11.27 +/- 21.39), whereas that to phytohaemagglutinin (PHA) was reduced (54.80 +/- 56.26 compared with 111.70 +/- 70.61). Both these results were statistically significant (P less than 0.01); they do not, however, appear to be due to a quantitative defect in T-cell numbers, as both total peripheral lymphocytes (3332 +/- 1450 cells/microliters compared with 2447 +/- 531 cells/microliters) and T-cells (2652 +/- 1128 cells/microliters compared with 1908 +/- 386 cells/microliters) were significantly elevated in the patients (P less than 0.01). When these tests were repeated two months later in 12 cured patients they were found to be comparable to those of the control subjects. The results indicate that there is a transient cellular sensitization to amoebic antigen together with an increase in T-lymphocytes and an impairment of the mitogenic response of lymphocytes to PHA. The significance of these findings is discussed.

Serodiagnosis of invasive amebiasis using a recombinant Entamoeba histolytica antigen-based ELISA.

Amebic serology remains a critical component in the diagnosis of invasive amebiasis. A recombinant serine rich-Entamoeba histolytica-protein/maltose binding protein (SREHP/MBP) fusion protein was evaluated as the target antigen in an ELISA test to detect acute invasive amebiasis. Retrospective analysis of 65 serum samples from patients with amebic liver abscess and 40 asymptomatic control patients showed the SREHP/MBP ELISA test had a sensitivity of 74% and specificity of only 55% for the diagnosis of amebic liver abscess. This study was repeated under identical conditions using a purified recombinant SREHP cleaved from the MBP component. The purified recombinant SREHP-based ELISA had a sensitivity of 79% and specificity of 87%. Our data suggest a purified recombinant SREHP-based ELISA could prove useful in the serodiagnosis of acute invasive amebiasis.

Intermediate host antigens associated with the cercariae of Schistosoma haematobium.

Bulinus (Physopsis) africanus antigens were shown to be associated with the cercarial glycocalyx of Schistosoma haematobium using immunofluorescence and the Cercarienhüllen Reaktion. It is proposed that this snail antigen may sensitise the definitive host and that resistance to further invasion by cercariae could be induced in this manner.

Differentiation of Entamoeba histolytica and Entamoeba dispar infections.

In this article, Terry jackson and Jonathan Ravdin briefly review the latest information on monoclonal antibody-based ELISAs that use antigen capture as a tool in the differential detection of human infection with Entamoeba histolytica and E. dispar. Current technology of culture and isoenzyme analysis is not widely available, is cumbersome and too time-consuming. A further potential benefit of antigen detection tests is that they can be used to monitor the efficacy of therapy; this is a shortcoming of serological tests owing to the persistence of the antibody response after successful treatment.

Frequency distribution of Entamoeba histolytica zymodemes in a rural South African population.

To determine the prevalence of Entamoeba histolytica and its zymodeme distribution pattern, isoenzyme electrophoresis was performed on isolates of the parasite from 1381 individuals of the population of a semirural endemic area south of Durban. About 10% of the population was found to harbour E histolytica. The zymodeme distribution was similar to that described in other surveys but two new zymodemes were observed, a non-pathogenic zymodeme XIX and a pathogenic zymodeme XX. Non-pathogenic zymodemes were more frequent in females than in males. In apparently healthy individuals there was a 1% prevalence of pathogenic E histolytica; the distribution was the same in males and females even though invasive amoebiasis was far more common in males. Symptoms of amoebic colitis subsequently developed in only 2 of the 14 symptomless individuals from whom pathogenic zymodemes of E histolytica were isolated.

A longitudinal study of asymptomatic carriers of pathogenic zymodemes of Entamoeba histolytica.

The value of treating subjects who pass Entamoeba histolytica cysts in their faeces is currently in question. In the endemic Durban area iso-enzyme electrophoresis of E. histolytica isolates indicated that 1% of the asymptomatic population are infected with pathogenic zymodemes. The outcome of these potentially invasive infections was established by means of a longitudinal survey. All subjects had strongly positive serological responses--10% of them developed amoebic colitis while the rest remained asymptomatic and spontaneous cure occurred within 1 year. Infections with pathogenic zymodemes occurred in family units and closely related individuals. Since they were cyst passers, good evidence was obtained for the existence of a carrier state for pathogenic E. histolytica. The value of sero-epidemiological surveys in determining the prevalence of pathogenic E. histolytica in a community was highlighted. Treatment of asymptomatic carriers with pathogenic zymodemes of E. histolytica is as essential as treatment of patients with invasive amoebiasis, and in both cases all contacts of affected individuals must also be treated if they are found to harbour pathogenic zymodemes of E. histolytica in order to ensure control of disease transmission.

Asymptomatic intestinal colonization by pathogenic Entamoeba histolytica in amebic liver abscess: prevalence, response to therapy, and pathogenic potential.

Since the application of isoenzyme electrophoresis to the study of Entamoeba histolytica, the prevalence and natural history of asymptomatic intestinal colonization in patients with amebic liver abscess (ALA) has not been addressed. We prospectively evaluated this enteric phase in 50 patients with ALA, using two dosage regimens of metronidazole. The overall prevalence of asymptomatic colonization was 72% (36/50). All these isolates, without exception, proved to express pathogenic zymodemes. Despite a 100% clinical response of the hepatic lesions, failure to eradicate the organism from the bowel occurred in 20 of these 36 subjects. During longitudinal posttreatment surveillance, three carriers returned with second bouts of invasive disease: one with dysentery and two with liver abscesses. Thus, in patients with ALA, there is a high prevalence of intestinal colonization with exclusively pathogenic strains, and treatment with metronidazole frequently results in a continued carrier state. These carriers have a propensity for developing recurrent invasive disease and constitute a public health hazard.

Evaluation of three serological tests for the detection of antiamebic antibodies applied to sera of patients from an area endemic for amebiasis.

Two enzyme immuno assays based on a single recombinant Entamoeba histolytica antigen (P1-EIA) or soluble E. histolytica extract (SA-EIA) as well as a latex agglutination test using an E. histolytica membrane fraction (M-LA) were evaluated for its use to detect anti-amebic serum antibodies in patients from Durban, South Africa, an area endemic for amebiasis. In a previous study, all three test systems were found to be reliable in terms of sensitivity and specificity when applied to sera of European individuals. By analysing a total of 167 serum samples of patients from the Durban area, suffering from invasive amebiasis (n = 76) or miscellaneous diseases unrelated to E. histolytica infection (n = 91), the present study revealed sensitivity for the detection of anti-amebic antibodies of 97.4% for SA-EIA, 86.8% for P1-EIA and 96.1% for M-LA, respectively. Specificity was high for P1-EIA (96.7%) and M-LA (92.3%) but substantially lower for SA-EIA (62.6%). In addition, antibody responses to the recombinant P1 antigen were analysed in 16 patients with amebic liver abscess before and after anti-amebic treatment. The results indicated that most of the patients lost their specific antibody response within 7 month of follow up. Therefore, P1-EIA seems to be a valuable test for distinguishing between present and past E. histolytica infections.