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Thesis (Ph.D.)—Boston University / Endamoeba histolytica, the etiological agent of amoebiasis, was first cultivated in the presence of associated bacteria by Boeck and Drbohlav (Am. J. Hyg., 5: 371-407, 1925). Since that time numerous workers have attempted to culture the amebas free of bacteria but have been unsuccessful. The majority of studies on the cultural requirements of this protozoan has been complicated by the presence of more than one species of bacteria. Therefore, the nutritional studies on E. histolytica have been very meager. A pure culture of this organism is essential to the elucidation of its metabolism, physiology, and immunology. It was the purpose of this investigation to identify growth factors required by the amebas so that a pure culture may become available. In order to control the associated bacteria during the assay of growth factors, penicillin and streptomycin were added to the culture medium in high concentrations. This concentration of the antibiotics was shown to sterilize the bacterial flora yet have no detrimental effect on the amebas. Many compounds were tested for their ameba-growth-promoting activity under bacteria free conditions. The test medium consisted of egg slants over-layed with normal horse serum in Ringer solution, dextrose, sodium thioglycollate, rice starch, antibiotics and a vaspar anaerobic seal [TRUNCATED]

A crude extract of axenically grown amoebae was used as antigen in order to develop an AB microELISA for the detection and quantitation of E. histolytica-specific IgG antibodies. This ELISA was used to screen individual sera of patients suffering from invasive amoebiasis (n=47)and control individuals(n=33). Significant titers of E-histolytica- specific IgG antibodies were present only in sera of patients suffering from invasive amoebiasis. The AB-microELISA had a sensitivity of 98% and a specificity of 97%. Immunoblot analysis of the crude E. histolytica extract indicated the presence of several antigenic proteins. One of the common antigenic proteins recognized by the individual patients' sera had a molecular weight of 160.170 kDa Sera of five patienta with high titers of E. histotytica-specific IgG antibodies were used to prepare a serum pool. This pooled serum was used for immunoscreening of an E. histolyitca cDNA expression library prepared in the phage vector l ZAP-II. A strongly immunoreactive phage done was identified, from which he recombinant phagemid was released by in vivo excision for characterization of the cDNA insert.

Amebiasis is a severe infection caused by the protozoan parasite Entamoeba histolytica (Schaudinn, 1903). This species is morphologically indistinguishable from Entamoeba dispar (Brumpt, 1925), considered to be non-pathogenic and unable to cause invasive illness. The differentiation between these two species by immunological and molecular biology techniques is important to determine the epidemiological situation, therapeutic intervention, and profilaxis of invasive amebiasis. The aim of this study was to discriminate infection caused by E. histolytica and E.dispar in schoolchildren population in the city of Maceió, Alagoas. A cross-sectional study was carried out among 1,003 students in state public schools. Stool examination by sedimentation technique was conducted in order to evaluate frequency of enteroparasites, and for screening of students infected with E. histolytica/E. dispar complex. Positive exams for Entamoeba cysts were further submitted to antigen detection assay by enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR), for specific diagnosis of the infection. Frequency of intestinal parasites among the examined population was high, being detected 54.8% (550/1,003) students harbouring at least one species of helminth or protozoan parasites. Most of the students families (57.5%), belonged to class D economic level according to the Brazilian Economic Classification Criteria. Of the total assessed 83.1% had an income ≤ 1 minimum wage and lived in households with 2-5 residents. Polymerase chain reaction (PCR) was standardized extracting DNA from the stool using Stool Kit PSP (Invitek ®). The primers selected by in silico analysis based on virtual performance for amplification of E. histolytica and E. dispar were p11plus/p12plus and p13plus/p14plus, respectively. The prevalence of amebiasis among schoolchildren diagnosed by ELISA was 3.0% (30/1,003) and by PCR 2.8% (28/1,003), but the difference was not statistically significant (p> 0.05). The overall prevalence of E. dispar in schoolchildren was 5.0% (50/1,003). Both techniques (ELISA and PCR) were appropriate for differential diagnosis of E. histolytica and E. dispar. However, ELISA has advantages such as its rapid and easy execution. The results indicates the importance of specific diagnostic techniques for amoebiasis detection in the population. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A amebíase é uma infecção severa que tem como agente etiológico o protozoário Entamoeba histolytica (Schaudinn, 1903). Esta espécie é morfologicamente indistinguível da Entamoeba dispar (Brumpt, 1925), considerada apatogênica, incapaz de provocar a forma invasiva da doença. A diferenciação entre as duas espécies através de técnicas de diagnóstico imunológico e de biologia molecular, é fundamental para determinação da situação epidemiológica da amebíase, estabelecimento da conduta terapêutica adequada, e consequentemente prevenção da doença invasiva. O objetivo do presente estudo foi discriminar a infecção causada pela E. histolytica e E. dispar na população de escolares da cidade de Maceió, Alagoas. Para tal, realizou-se um estudo de corte transversal, com 1.003 estudantes da rede pública estadual de ensino da cidade. Foi realizado exame de fezes, pelo método de sedimentação espontânea, para avaliação da frequência das parasitoses intestinais, e para a triagem dos escolares infectados pelo complexo E. histolytica/E. dispar. Os exames positivos para cistos de Entamoeba foram posteriormente submetidos à pesquisa de antígeno pelo ensaio imunoenzimático (ELISA) e reação em cadeia da polimerase (PCR), para o diagnóstico específico da infecção. A frequência de enteroparasitos entre os examinados foi elevada, sendo identificados 54,8% (550/1.003) escolares infectados por pelo menos uma espécie de helminto ou protozoário. A reação em cadeia da polimerase (PCR) foi padronizada utilizando para extração do DNA das fezes o Kit Stool PSP (Invitek®). Os iniciadores selecionados através da análise in silico pelo bom desempenho virtual para amplificação da E. histolytica e da E. dispar, foram p11plus/p12plus e p13plus/p14plus, respectivamente. A prevalência da amebíase nos escolares diagnosticada pelo ELISA foi de 3,0% (30/1.003) e na PCR 2,8% (28/1.003), não existindo diferença significativa entre as percentagens de infectados (p>0,05). A prevalência geral da E. dispar nos escolares foi de 5,0% (50/1.003). Ambas as técnicas (ELISA e PCR) se mostraram adequadas para diagnóstico diferencial da E. histolytica e E. dispar. No entanto, o ELISA apresenta a vantagem ser de rápida e simples execução. Os resultados apresentados indicam a necessidade da utilização de técnicas de diagnóstico específico para detecção de casos amebíase na população.

Amoebic dysenteri is a problem in developing countries and is caused by Entamoeba histolytica (E. histolytica) with symptoms such as diarrhea, vomiting and in worse case extra intestinal manifestation. Currently there are difficulties to diagnose E. histolytica infections in developing countries because PCR requires advanced and expensive and microscopy cannot distinguish E. histolytica from other harmless species of amoebas. The aim of this study was therefore to develop loop-mediated isothermal amplification (LAMP), which is similar to PCR but is performed at a single temperature and amplifies the target gene in less than an hour. LAMP was also compared to real time PCR. With a commercial kit, DNA were extracted from cultivated trophozoites and for the LAMP reaction, a colorimetric mastermix and six primers were used designed from 18S small subunit ribosomal RNA gene. With phenol red positive LAMP reactions showed a color change from pink to yellow and negative LAMP reactions remained pink. The sensitivity of LAMP for detection of E. histolytica was determined to be 80 pg/µl, which was ten times less sensitive than real time-PCR. The method was also shown to work on trophozoites with no DNA extraction and no non-specific amplifications were seen with DNA from G. lamblia, which showed some specificity. LAMP proved to be sensitive and easy to work with, but requires tightly closed tubes to avoid contamination and false positive results. To develop and evaluate the method LAMP for detection of E. histolytica, more studies are needed, including clinical samples and optimization.

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