Use of an enzyme-linked immunosorbent assay to detect anti-adherence protein antibodies in sera of patients with invasive amebiasis in Cairo, Egypt.

We used enzyme-linked immunosorbent assay (ELISA) to detect IgG antibodies to the Entamoeba histolytica galactose-inhibitable adherence protein in the sera of 50 uninfected controls, 50 cases with asymptomatic cyst passage, 100 patients with amebic colitis, and six patients with amebic liver abscess from Cairo, Egypt, and in 50 healthy controls from the United States. When the mean + 3 SD value above that of the controls from the United States was used as a criterion for a positive ELISA result, 100% of those with invasive amebiasis, 80% of those with asymptomatic infection, and 64% of the Egyptian controls had anti-adherence protein antibodies. However, when the mean + 2 SD value of Egyptian control sera (optical density = 0.094) was used as the criterion for positivity, 33 (89%) of 37 sera from individuals with invasive amebiasis having symptoms for at least one week were antibody positive, in contrast to only 12% of asymptomatic cyst passers (P < 0.01). In a highly endemic area such as Cairo, Egypt, detection of serum anti-adherence protein antibodies by ELISA may have greatest diagnostic use in patients with symptomatic invasive amebiasis of greater than one week duration.     

Association between parasitic intestinal infections and acute or chronic diarrhoea in HIV-infected patients in Caracas,Venezuela

A cross sectional survey was conducted to determine the association between enteric parasites and diarrhoea in HIV-infected adults in Caracas. Three hundred and four patients were evaluated: 104 had acute diarrhoea, 113 chronic diarrhoea and 87 were controls. Isopora belli infection was associated with acute (P = 0.022) and chronic diarrhoea (P = 0.003), Entamoeba histolytica/dispar infection was also associated with both acute (P = 0.015) and chronic diarrhoea (P = 0.017). Strongyloides stercoralis (P = 0.003), and Cryptosporidium parvum (P = 0.017) infections were associated mainly with chronic episodes. Weight loss (P < 0.001), a non-infectious factor investigated, was significantly associated with diarrhoea. Eosinophilia, a laboratory parameter studied, was found to be associated with strongyloidiasis (P = 0.001), giardiasis (P = 0.001) and isoporiasis (P = 0.003). In summary, the presence of enteric parasites in HIV-infected patients from tropical urban areas with diarrhoea, with or without significant weight loss, must be considered. Similarly, eosinophilia might suggest parasitic infection in these patients.     

Immunohistochemical characterization of human fulminant amoebic colitis

In cases of fulminant amoebic colitis we have determined the interactions between Entamoeba histolytica trophozoites and immune cells in order to better understand the pathophysiology of amoebic colitis. Eleven specimens of amoebic colitis and five specimens of colon without amoebic lesions were studied. Trophozoites and immune cells were located by topographic stains, histochemistry and immunohistochemistry. Trophozoites were seen in both damaged and undamaged areas of the colonic mucosa. Specimens of fulminant amoebic colitis showed: (a) an increase in IgA+, IgG+ B cells and neutrophils; (b) a reduction in IgM+ B cells, CD8+ T cells, macrophages, eosinophils and mast cells; and (c) no change in the number of NK and CD4+ T cells. The cellular infiltrate in amoebic colitis may represent the combined effects of amoebic monocyte locomotion inhibitory factor and switching of IgM+ B cells to IgG+ and IgA+ plasma cells, induced by amoebic antigens. Tissue damage in the absence of trophozoites may result from ischaemia or host immune responses.     

Entamoeba histolytica infection in men who have sex with men

Entamoeba histolytica infection (amoebiasis) is the second leading cause of death from parasitic diseases. Epidemiological studies from developed countries have reported an increasing prevalence of amoebiasis and of invasive infections, such as amoebic colitis, among men who have sex with men (MSM) who engage in oral-anal sex. Although most infections with E histolytica are asymptomatic, clinical manifestations of invasive amoebiasis mainly include amoebic colitis and amoebic liver abscess, which are associated with substantial morbidity and medical cost. Laboratory diagnosis of amoebiasis should be based on detection of E histolytica by use of tests with high sensitivity and specificity, such as specific amoebic-antigen or PCR-based assays. Microscopy used in routine clinical laboratories is not sensitive or specific enough for detection of E histolytica. Metronidazole or tinidazole remains the mainstay of treatment for invasive amoebiasis, followed by treatment with luminal agents to prevent relapse and transmission of E histolytica to sexual partners or close contacts.     

HLA antigens associated to amoebic abscess of the liver in Mexican mestizos

Worldwide prevalence of amoebiasis is estimated at 4 x 10(8) cases/year, yet only one of about 300 individuals harbouring Entamoeba histolytica suffers tissue invasion and these cases are mostly concentrated in certain areas of Asia, Africa and Latin America. Patients with amoebic abscess of the liver (AAL) represent only a small fraction of that. These contrasting figures have been tentatively explained on the one hand through variations in sex, immunocompetence, nutritional and other socioeconomic features of the host, and on the other hand through differences in parasite virulence. In order to explore a possible association between the major histocompatibility complex (MHC) and AAL susceptibility, we studied the HLA profile in 31 Mexican mestizos with AAL and compared it to race and socioeconomically matched controls. Mexican mestizo patients with AAL revealed a significant increase in HLA-Bw16 and HLA-DR3 which could suggest an HLA-related susceptibility to liver invasion by E. histolytica.     

Fundamental studies on serological diagnosis of amoebiasis. 2. Application of antigen of Entamoeba histolytica for complement fixation test to counterimmunoelectrophoresis; and clinical relevance of serological tests

Antigen of Entamoeba histolytica for complement fixation (CF) test was applied to counterimmunoelectrophoresis (CIE), and the result was compared with that of CF test and enzyme-linked immunosorbent assay (ELISA) in regard to sensitivity and specificity. Besides, we studied antibody titers to E. histolytica in sera from acute and convalescent patients, and from patients with amoebic colitis and amoebic liver abscess. Sera used were from patients with amoebic colitis, amoebic liver abscess and uninfected control subjects. The CIE was less sensitive than the CF test and ELISA. Most CIE-negative samples had low CF and ELISA titers, but a few samples had higher CF and ELISA titers. On the other hand, all uninfected controls were negative by CIE and ELISA, and 98% were negative by the CF test. The level of antibodies tended to decrease with time after clinical recovery, but CF test and ELISA results remained positive in all patients 6 months after clinical cure. CIE converted negative in only one patient after 6 months. The level of antibodies of CF test and ELISA was significantly higher in amoebic liver abscess than amoebic colitis. These results suggested that all these three methods were so specific, and antibody titers by the CF test and ELISA were well correlated to clinical manifestations. The CIE with CF-antigen was less sensitive as compared with CF test or ELISA.     

多聚酶链反应分析溶组织内阿米巴的致病性

从急性阿米巴痢疾患者粪便中分离所得的溶组织内阿米巴虫株SH-3、SH-6、SH-8的DNA和包囊携带者粪便中分离所得的溶组织内阿米巴 SH-5、SH-7的 DNA应用多聚酶链反应技术扩增30个周期后作琼脂糖凝胶电泳分析,发现致病性引物 P_(11)、P_(12)可使SH-8虫株的DNA增殖;而非致病性引物P_(13)、P_(14)可使SH-3、SH-5、SH-6和SH-7虫株的DNA增殖。另外,酶株群分析和单克隆抗体反应也显示SH-8为致病性虫株,而另4株虫株均属于非致病性虫株,与多聚酶链反应的结果相一致。提示,致病性虫株与非致病性虫株的基因有区别,多聚酶链反应对溶组织内阿米巴进行基因分析是一种敏感和特异的新方法。     

Amoebiasis

Amoebiasis is the second leading cause of death from parasitic disease worldwide. The causative protozoan parasite, Entamoeba histolytica, is a potent pathogen. Secreting proteinases that dissolve host tissues, killing host cells on contact, and engulfing red blood cells, E histolytica trophozoites invade the intestinal mucosa, causing amoebic colitis. In some cases amoebas breach the mucosal barrier and travel through the portal circulation to the liver, where they cause abscesses consisting of a few E histolytica trophozoites surrounding dead and dying hepatocytes and liquefied cellular debris. Amoebic liver abscesses grow inexorably and, at one time, were almost always fatal, but now even large abscesses can be cured by one dose of antibiotic. Evidence that what we thought was a single species based on morphology is, in fact, two genetically distinct species--now termed Entamoeba histolytica (the pathogen) and Entamoeba dispar (a commensal)--has turned conventional wisdom about the epidemiology and diagnosis of amoebiasis upside down. New models of disease have linked E histolytica induction of intestinal inflammation and hepatocyte programmed cell death to the pathogenesis of amoebic colitis and amoebic liver abscess.     

Comparison of conventional and immunofluorescent techniques for the detection of Entamoeba histolytica in rectal biopsies

Two comparative studies of six staining techniques in detection of Entamoeba histolytica in rectal biopsies were performed. The techniques evaluated were direct and indirect fluorescent antibody technique, and four conventional stains: PAS, H & E, Trichrome, and PTAH). On ethanol-fixed tissue, the direct fluorescent antibody technique was the most sensitive, detecting amoebas in 84% of rectal biopsies from 19 aspirate-positive dysentery patients. PAS staining was specific, almost as sensitive, and amoebas could be rapidly detected. The other three conventional methods, however, detected amoebas in only 53--58% of the aspirate-positive patients and required considerable efforts to find the amoebas. The indirect fluorescent antibody technique performed on ethanol-fixed tissues was comparable to the direct technique. The same technique used on formalin-fixed tissues had high background staining, but amoebas could still be detected more efficiently than with conventional staining. Following 20 hr or more treatment with metronidazole, no patient had amoebas detectable in their rectal biopsies by any technique used. Patients with amoebic dysentery and necrotic ulcers on protoscopy had amoebas detected more frequently in rectal biopsy than did patients with mild disease as determined by proctoscopy. Entamoeba histolytica in rectal biopsies can be detected most easily in ethanol-fixed tissue stained with either a fluorescent antibody or with PAS. If amoebas are not detected in the rectal biopsy, amoebic colitis cannot be excluded.     

Regulation of the inflammatory immune response by the cytokine/chemokine network in amoebiasis

Amoebiasis is caused by the protozoa Entamoeba histolytica and persists as one of the leading parasitic diseases affecting millions worldwide. This parasite invades the intestinal mucosa, causing amoebic colitis and ulcers. It may also spread to other organs, mainly the liver, causing amoebic liver abscess (ALA). Current research efforts have focused on the development of specific diagnostic tests and animal models searching for a better understanding of the complex physiopathology of this disease. Analysis of the inflammatory immune response during intestinal amoebiasis in both human disease and animal murine models has revealed an important regulatory role for chemokines and cytokines. Recruitment and activation of inflammatory cells can also be modulated by specific protease-mediated cleavage of cytokines and by secreted amoebic factors such as amoebapores and monocyte locomotion inhibitory factor (MLIF). Unlike intestinal amoebiasis, analysis of the immune response in ALA has mainly been done in the hamster model. This has limited our information regarding the immune response during this phase of the disease. However, even with these limitations, several Th1/2 cytokines, such as IL-6 and IL-4, and regulatory cytokines, like IL-10 and TGFbeta, have been associated to the development of this disease.     

The interaction of human T-lymphocytes and Entamoeba histolytica: killing of virulent amoebae by lectin-dependent lymphocytes

Clinical and experimental studies indicate that following invasive disease due to Entamoeba histolytica, development of human cell-mediated immune mechanisms may provide protective immunity. Activated, human monocyte-derived macrophages in vitro can kill virulent axaenic amoebic trophozoites. This study describes the interaction of lectin-stimulated T-lymphocytes and E. histolytica trophozoites (virulent strain HM1-IMSS). Amoebae progressively killed unstimulated nonimmune T-lymphocytes over 18 h incubation with no effect on amoebic viability. T-lymphocytes, stimulated with phytohaemagglutinin (PHA), were progressively cytotoxic for virulent HMI amoebae over 18 h incubation, but were also reduced in viability themselves. Lymphocyte cytotoxicity for amoebae was absent if PHA was removed before or added only during the assay. PHA-stimulated T-lymphocytes killed amoebae at cell ratios of lymphocytes to amoebae as low as 50:1 and cytotoxicity was antibody-independent. PHA-stimulated T-lymphocytes, depleted of T8-bearing cells by complement-mediated lysis, were unable to kill amoebae. Adherence of PHA-stimulated T-lymphocytes to amoebae was greater than with unstimulated T-lymphocytes. Inhibition of the amoebic adherence lectin with N-acetyl-D-galactosamine decreased lymphocyte-amoebic adherence and resulted in increased lymphocyte amoebicidal activity and lymphocyte survival. Suspension of amoebae with or without adherent PHA-stimulated T-lymphocytes in a 10% dextran solution indicated that cytotoxicity was contact dependent. In summary, PHA-stimulated T-lymphocytes of the T8-phenotype can kill virulent axaenic E. histolytica trophozoites through a contact-dependent, antibody-independent mechanism.     

Macrophage requirement for host defence against experimental hepatic amoebiasis in the hamster

The role of macrophages in hepatic amoebiasis in hamsters has been investigated by means of their treatment with bacille Calmette-Guérin (BCG) for activation, and with silica for elimination of these cells. Silica-treated animals inoculated intrahepatically with 1 x 10(5) trophozoites of Entamoeba histolytica developed amoebic abscesses in the liver and more metastases to other organs than control animals, and this effect was silica-dose-dependent. In contrast, BCG-treated animals developed significantly smaller abscesses in the liver and fewer metastatic foci. These data suggest that macrophages are involved in host defence against the establishment of amoebic liver abscess and metastatic dissemination of amoebae.     

Antigenicity and immunogenicity of phage library-selected peptide mimics of the major surface proteophosphoglycan antigens of Entamoeba histolytica

Entamoeba histolytica is the protozoan parasite responsible for intestinal amoebiasis and amoebic liver abscess, which cause significant morbidity and mortality in many countries of the world. Proteophosphoglycans (PPGs, also known as lipophosphoglycans, LPGs, or lipopeptidophosphoglycans, LPPGs) represent dominant surface components of E. histolytica. Passive immunization with a monoclonal antibody (EH5) directed against these components protected SCID mice from amoebic liver abscess, so PPGs might be regarded as vaccine candidates; however, their structure is very complex and only known in part. They are glycosylphosphatidylinositol-linked polypeptides of unknown sequence carrying glycan side-chains linked to serine residues via phosphodiester bonds. In order to identify peptide mimics of the E. histolytica PPG antigens, we screened six different phage-displayed random peptide libraries with the antibody EH5. Various peptide mimics of different length were identified and, in all the peptides, a distinct consensus sequence Gly-Thr-His-Pro-X-Leu could be identified. The phages strongly bound to the antibody, and the natural antigen inhibited binding of the phages to antibody EH5. In addition, several of the phages induced a significant immunoglobulin G response against amoebic antigens in immunized mice.     

Cellulose acetate precipitation test in sero-diagnosis of amoebiasis

Cellulose acetate precipitation (CAP) test for the detection of antibody against Entamoeba histolytica using the axenic antigen, was performed on the 127 serum samples obtained from patients with amoebic liver abscess (14), amoebic hepatitis (21), amoebic dysentry (11), amoebic colitis (31), other parasitic infestations (25) and normal individuals (25). The percent positivity was 100, 95.23, 90.9, 67.74 and 16 and 12 respectively whereas the corresponding figures for the indirect immunoflourescence (IFAT) test were 100, 100, 100, 74.19, 12 and 8 respectively. Although CAP is not as good a test as IFAT, yet it can be recommended for routine testing due to its sensitivity, speed of performance and applicability to a single serum sample.     

Immunoprecipitation studies with biotinylated Entamoeba histolytica antigens

The biotinylation of protein antigens for use in immunoprecipitation studies with parasite antigens is described. This technique was standardized to produce maximal labelling without compromising either the viability of the parasite or the antigenicity of labelled proteins. Live axenic Entamoeba histolytica trophozoites were optimally labelled by incubation of 10(7) parasites in 1 ml phosphate-buffered saline for 20 min at 37 degrees C with a final concentration of 10 mM N-hydroxy succinimido-biotin (NHS-B). Examination of antigens recognized by immunoglobulin from convalescent amoebic liver abscess patients and precipitated by protein A-Sepharose showed a general increase in immune response to amoebic antigens with time following treatment. Mass ratio mol. wts of immunoprecipitated antigens concur with those presented by other authors using alternate technology, in addition to recognizing antigens previously not identified by human sera. We therefore recommend biotinylation as a viable alternative to radiolabelling techniques for the study of parasite antigens and the humoral immune responses raised against them.     

Increased leucocyte histamine release by Entamoeba histolytica antigen in patients with amoebic abscess of the liver

Leucocytes (basophils) from non-atopic adult subjects living in an area highly endemic with Entamoeba histolytica release histamine in a dose-dependent fashion upon in vitro exposure to an antigen of axenically grown E. histolytica (histolyticin). Leucocytes of patients with acute amoebic liver abscess were significantly more sensitive to this antigen than leucocytes of control subjects, including patients that had recovered from amoebic liver abscess. By comparison Concanavalin-A induced histamine release found in patients with amoebic liver abscess and healthy controls suggest an immunological mechanism for histolyticin induced in vitro histamine release. This is also suggested by the inability of histolyticin to release histamine from leucocytes of healthy newborn infants and the significant fall in sensitivity to histolyticin following incubation of leucocytes in acid pH. Histamine and other mediators may contribute locally to the early intense inflammatory reaction observed in tissue invasion by E. histolytica.     

Effect of ion channel inhibitors on the cytopathogenicity of Entamoeba histolytica

Entamoeba histolytica (axenic strain HM1-IMSS), a cytolytic enteric pathogen, kills target Chinese hamster ovary cells in two discrete steps: (1) carbohydrate-specific adherence of amoebae to target cells, followed by (2) cytolysis of adherent target cells. Both steps require intact amoebic microfilament function. The effects of the fast Na+ channel blocker tetrodotoxin and the slow Na+-Ca++ channel blockers verapamil (10(-5) M) and bepridil (10(-5) M) on amoebic killing were evaluated. Verapamil and bepridil both decreased amoebic killing (P less than 0.001); tetrodotoxin had no effect. Bepridil, but not verapamil, inhibited amoebic adherence at 37 C (P less than 0.001). Both verapamil and bepridil inhibited amoebic lysis of cells after adherence occurred (P less than 0.001). Verapamil protected target cells from lysis by amoebae (P less than 0.005), whereas bepridil reduced the capacity of amoebae to kill Chinese hamster ovary cells (P less than 0.001). These findings suggest that changes in transmembrane ion flux in both the amoeba and the target cell are involved in amoebic killing of target cells.     

Comparison of antigen-capture ELISA to stool-culture methods for the detection of asymptomatic Entamoeba species infection in Kafer Daoud, Egypt

We performed a prospective field study in the village of Kafer Daoud in Menofia, Egypt to compare the fecal culture method with enzyme linked immuno assay (ELISA) for detection of 170 kDa lectin antigen in feces for diagnosis of asymptomatic Entamoeba histolytica and Entamoeba dispar infection. All subjects with E. histolytica or E. dispar infection detected by culture also had positive ELISA for amebic antigen in their feces and an additional 57 Entameoba infections missed by culture were detected by ELISA (P < 0.001 compared to culture). The presence of fecal anti-lectin IgA antibodies and serum anti-LC3 (recombinant cysteine-rich lectin protein) IgG antibodies were positive predictors for E. histolytica infection (P < 0.03). Of interest, infection with Trichomonas hominis but not Blastocystis hominis was positively associated with E. histolytica infection (P < 0.05). In conclusion, ELISA for detection of fecal lectin antigen is a more sensitive method than fecal culture for detecting asymptomatic E. histolytica infection.     

兰州市慢性病患者隐孢子虫和溶组织阿米巴感染流行病学调查结果分析

目的了解兰州市慢性病（主要为高血压、糖尿病和恶性肿瘤）人群隐孢子虫和溶组织阿米巴的感染状况。方法在兰州市收集高血压、糖尿病、恶性肿瘤及非慢性病患者等的粪便,分别采用改良抗酸染色法和碘染色法,检测粪便中的隐孢子虫卵囊和溶组织阿米巴包囊。结果在600例粪便中,隐孢子虫和溶组织阿米巴感染率分别为4．17％和4．50％,感染率差异无统计学意义（χ2=0．080,P〉0．05）;男性感染率分别为4．67％和4．33％,女性感染率分别为3．67％和3．oO％;〈10岁者隐孢子虫和溶组织阿米巴的感染率最高,分别为6．00％和7．00％;糖尿病和恶性肿瘤组隐孢子虫的感染率分别为7．00％和10．。0％,与对照组比较差异有统计学意义（χ2=4．178,7．639;P〈0．05）;糖尿病和恶性肿瘤组溶组织内阿米巴的感染率分别为9．00％和8．33％,与对照组比较差异有统计学意义（χ2=6．866,4．108;P〈0．05）。结论兰州市隐孢子虫和溶组织阿米巴儿童和慢性病患者人群感染率较高,可能与该人群健康状况、免疫力和卫生习惯等密切相关。