Emma I. Melendro

The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica

Formalin fixation is the most common storage, transportation and preservation method for stool samples. However, fixation dramatically reduces our ability to extract from stool samples DNA that is a suitable template for polymerase chain reaction (PCR)-based diagnostic tests. In this study we evaluated the effects of formalin concentration and of the time stored in fixative on the success of PCR amplification. We found that the deleterious effects of formalin are both time and concentration dependent and may result from fragmentation of fixed DNA during its purification.

Prevalence of Entamoeba histolytica and Entamoeba dispar in a Highly Endemic Rural Population

Amebiasis is caused by the protozoan Entamoeba histolytica. This disease, or the asymptomatic infection, is associated with poor socioeconomic conditions, malnutrition, and poor hygiene behavior, all common in developing countries. There is evidence of the existence of two species of quadrinucleated amebas in the human population ( E. histolytica and Entamoeba dispar ) with different pathogenic capabilities, making necessary the reevaluation of the actual frequency of infection and disease, because is a tendency to overestimate it in endemic areas where other intestinal infections causative of dysentery or bloody diarrhea are found. The opposite is frequent in nonendemic areas, where the presence of amebas is overlooked in stool microscopic examination. In the present work, we studied the prevalence of E. histolytica or E. dispar infection in the rural community of Coahuixtla, State of Morelos, Mexico. The selection of this community was made on the basis of the reported rate of new cases of intestinal amebiasis or amebic liver abscess in the state of Morelos (1). E. histolytica and E. dispar identification was performed through the molecular analysis of the cysts isolated directly from stool samples.

Entamoeba histolytica: antibody response to recent and past invasive events

Sero-epidemiological data from endemic amoebiasis areas are difficult to evaluate because the serology of individuals affected by an active process of Entamoeba histolytica tissue invasion is, at present, almost impossible to distinguish from that of individuals who have had an invasive event in the past. The present study compares serum antigenic recognition frequencies among three groups of individuals with different infective conditions: amoebic liver abscess patients; asymptomatic cyst passers; and individuals who have had amoebic liver abscess from one to three years before the study. Control groups consisted of Mexican and Canadian healthy adults. Western blots of E. histolytica membrane extract antigen were reacted with sera from the studied individuals, recognition frequency values were calculated and immunoplots of frequency differences were constructed. The results obtained suggest that the identification and purification of antigenic fractions, which are frequently recognized by sera of amoebic liver abscess patients (136, 132, 93, 70 and 62 kDa), or preferentially associated with past invasive events (144, 140 and 49 kDa), or related to the E. histolytica cyst passer condition (62 and 136 kDa), are important improvements in the use of serology for diagnosis and epidemiological studies in endemic areas of amoebiasis.

Western blot of Entamoeba histolytica antigenic fractions: reactivity analysis with sera from intestinal amoebiasis patients.

The reactivity of sera from acute-phase intestinal amoebiasis patients (two weeks evolution) was studied to determine which of the Entamoeba histolytica antigens are most frequently immunogenic. Sera were examined by means of immunoelectrotransferase assay using crude extract of HM1:IMSS E. histolytica trophozoites. Three populations of clinically healthy adults from Mexico, Canada and Germany, with no evidence of parasites in faeces, were used as controls. The frequency of antigen recognition was analysed. In ailing individuals, the bands of 23, 24, 26 and 51 kDa were recognized most frequently (65 and 60%) followed by the 62 kDa band (56%). The combination of some of these bands, namely 3.4, 4.1 and 6.7, with molecular weights of 62, 51 and 24 kDa, increased the recognition frequency of patients to 91.4%. These results constitute a first but important step towards the design of more accurate methods for the successful immunodiagnosis and epidemiology of acute intestinal amoebiasis.

Entamoeba histolytica: specific antigen recognized by a monoclonal antibody.

Specific antigenic determinants on the membrane surface of Entamoeba histolytica that distinguish it from other Entamoeba species were demonstrated. Evidence for these antigenic determinants was obtained with a monoclonal antibody to E. histolytica which showed not only specificity but also sensitivity as demonstrated in enzyme linked immunosorbent assay. Immunofluorescence microscopy showed that the monoclonal antibody recognized an epitope present on the membrane surface of E. histolytica trophozoites. The epitope detected by the monoclonal antibody was present in three components of different molecular weight. These components may have a common precursor or may be the result of enzymatic degradation under the conditions tested.

Resistance to Nocardia brasiliensis infection in mice immunized with either Nocardia or BCG.

Different vaccination procedures to increase the mecha nisms of host resistance to Nocardia brasiliensis were studied in mice. When mice were challenged in the footpad, 2×108N. brasiliensis 20 days after footpad inoculation with either viable or killed N. brasiliensis, the mice demonstrated significant resistance to infection when compared with noninfected and nonimmunized mice. The degree of resistance seems to be correlated with the delayed-type hypersensitivity response in the vaccinated animals. Vaccination with another acid-fast bacilli, BCG, afforded both a mild protection and low DTH reactivity. Antibody levels to Nocardia were similar in either Nocardia- or BCG- treated groups indicating that they do not play an important role in resistance to infection by N. brasiliensis.

Changes in Host Resistance Caused by Nocardia brasiliensis in Mice: Cross-Protection against Listeria monocytogenes

Listeria monocytogenes was used to study the rate of development, magnitude, and persistence of the antimicrobial resistance engendered by Nocardia brasiliensis infection in mice. The growth of Listeria in the liver and spleen was more effectively restricted in Nocardia-infected mice than in noninfected animals. The development of delayed-type hypersensitivity to the Nocardia antigen was closely correlated to the increased resistance to Listeria, suggesting that both properties are the consequence of a single immunological event. The antibacterial resistance was also demonstrated in vitro. The results of the foregoing studies indicate that the microbicidal ability of macrophages, very likely activated by cell-mediated immunity, in enhanced in mice infected with Nocardia.

Purification and Biochemical Characterization of Three Cysteine Proteases of Entamoeba histolytica with Potential Application in Epidemiologic Trials

Entamoeba histolytica is responsible for human invasive amebiasis. This disease has an endemic behavior in specific geographic areas throughout the world, and is commonly associated with population with low socioeconomic and educational conditions (1). It is in these endemic areas where the evaluation of prevalence of infection and disease through the detection of antiameba antibodies in serum and secretions is particularly valuable. Although there is an increasing number of new strategies to make available better antibody and antigen detection tests (2), the complexity of data analysis obtained in epidemiologic trials in highly endemic areas justifies the search for new antigens of E. histolytica with biological or molecular characteristics that may optimize antibody detection tests for use in field studies. In the present article, we describe the purification of three proteases of E. histolytica HM-1:IMSS that previously showed to be highly recognized (91.4%) by serum antibodies from patients with amebic dysentery (3), and in more than 95% of amebic liver abscess patients. Preliminary data suggest that antibody levels to these proteins decrease significantly after 6 months of treatment, which makes them attractive for the detection of new cases of invasive amebic disease.

B LYMPHOCYTE STIMULATION BY PARASITIC ORGANISMS

Publisher Summary This chapter discusses B-lymphocyte stimulation by parasitic organisms. After exposure to either antigens or mitogens, previously resting lymphocytes are stimulated to synthesize DNA and undergo numerous divisions that lead to the formation of clones of cells. Cells having an enlarged cytoplasm, which contains membranous structures that synthesize and actively secrete elevated quantities of immunoglobulin molecules, are developed in these clones. The term polyclonal mitogens is used to denote ligands that induce a large proportion of T- or B-lymphocyte populations to enter mitosis in vitro . Mitogens stimulate B cells to undergo two intracellular chain reactions: the induction of DNA synthesis and cell proliferation and the initiation of increased synthesis and active secretion of IgM. The degree of stimulation induced by different mitogens can vary. These substances, also known as polyclonal B cell activators (PBCAs), can stimulate B cells nonspecifically and directly. PBCAs activate B lymphocytes to perform the function for which they are genetically programmed and are able to carry out according to their state of differentiation.

ENTAMOEBA HISTOLYTICA ADHERENCE: INHIBITION BY IgA MONOCLONAL ANTIBODIES

Adhesion of Entamoeba histolytica to target cells in the host intestine, is the first of three consecutive steps (adhesion, cytolytic effect and phagocytosis) involved in the invasion of colonic tissues.1 The present study investigated the role of the local secretory immune response in the-interference with this early host-parasite relationship. IgA monoclonal anti-E. histolytica antibodies were produced. One of the clones obtained (F1P1D5) had been tested in its capacity to block the adhesion process in vitro with two different epithelial cells (MDCK and HT-29 cell lines), and in situ using colonic mucosa from BALB/c mice or gerbils (Meriones unquiculatum), which differ in susceptibility to Entamoeba experimental infection.