Entamoeba histolytica: fecal antigen capture immunoassay for the diagnosis of enteric amebiasis by a monoclonal antibody

Amebiasis continues to be of epidemiological importance in underdeveloped countries. Clinical diagnosis and epidemiological setting in a region are based on the fecal microscopic identification of cysts or trophozoites. This procedure requires well trained personnel, is laborious, of low sensitivity and frequently yields false-positives results. The present study was designed to develop an immuno-enzymatic fecal 96 kDa antigen capture test (COPROELISA-Eh) more sensitive and specific than microscopic diagnosis of amebiasis. Triplicates of 177 stool samples processed by the formol-ether concentration method, were defined as positive or negative by three experienced microscopic observers. Another aliquot was submitted to the antigen capture test by a monoclonal antibody against a specific membrane antigen of pathogenic strains of Entamoeba histolytica. Optical densities were interpreted as positive when they exceeded the mean value of negative samples plus two standard deviations. COPROELISA-Eh showed a 94.4% sensitivity, 98.3% specificity, 96.2% positive predictive value and 97.6% negative predictive value for the detection of E. histolytica in feces. COPROELISA-Eh is more sensitive and specific than microscopic examination, does not require specially trained personnel and allows the simultaneous processing of a large number of samples.

Laboratory diagnosis of amebiasis in a sample of students from southeastern Brazil and a comparison of microscopy with enzyme-linked immunosorbent assay for screening of infections with Entamoeba sp.

Introduction: Epidemiological studies on amebiasis have been reassessed since Entamoeba histolytica and E. dispar were first recognized as distinct species. Because the morphological similarity of these species renders microscopic diagnosis unreliable, additional tools are required to discriminate between Entamoeba species. The objectives of our study were to compare microscopy with ELISA kit (IVD®) results, to diagnose E. histolytica infection, and to determine the prevalence of amebiasis in a sample of students from southeastern Brazil. Methods: In this study, diagnosis was based on microscopy due to its capacity for revealing potential cysts/trophozoites and on two commercial kits for antigen detection in stool samples. Results: For 1,403 samples collected from students aged 6 to 14 years who were living in Divinópolis, Minas Gerais, Brazil, microscopy underestimated the number of individuals infected with E. histolytica/E. dispar (5.7% prevalence) compared with the ELISA kit (IVD®)-based diagnoses (15.7% for E. histolytica/E. dispar). A comparison of the ELISA (IVD®) and light microscopy results returned a 20% sensitivity, 97% specificity, low positive predictive value, and high negative predictive value for microscopy. An ELISA kit (TechLab®) that was specific for E. histolytica detected a 3.1% (43/1403) prevalence for E. histolytica infection. Conclusions: The ELISA kit (IVD®) can be used as an alternative screening tool. The high prevalence of E. histolytica infection detected in this study warrants the implementation of actions directed toward health promotion and preventive measures.

Does the eosinophil have a protective role in amebiasis?

While normal human eosinophils are destroyed in vitro by virulent Entamoeba histolytica, notwhistanding the presence of antibodies and complement, activated eosinophils promptly destroy the parasite although dying also at the end of the process. To study the possible in vivo participation of eosinophils in invasive amebiasis, we compared the induction of experimental amebic abscess of the liver (AAL) in gerbils (Meriones unguiculatus) previously made eosinophilic through Toxocara canis antigen injection and in normal control gerbils. After intraportal inoculation of 10(5) ameba trophozoites (6 and 24 hr), the ratio of gerbils with AAL, as well as the number and size of the microabscesses was comparable in eosinophilic and control gerbils. However, at 96 hr the number and size of the microabscesses were significanly smaller (p<0.05) in eosinophilic gerbils. On the other hand the actuarial AAL survival curve up to 45 days post-amebic inoculation was signficantly (p<0.05) shifted to the right in controls. These results suggest that antigen-induced eosinophilia may exert a protective effect against AAL in gerbils.

Prevalence and pathogenicity of Entamoeba histolytica in three different regions of Pernambuco, Northeast Brazil

Parasitological examinations were carried out on 663 individuals of three different cities of Pernambuco State, Northeastern Brazil: Recife, Palmares and Bodocó. The population from a drought area of Pernambuco State, Bodocó, was investigated for amoebiasis and compared with Recife, metropolitan city (about 1.3 million of inhabitants) and another inland community, Palmares, located inside of the sugar-cane plantation region of the State. No evidence of invasive strains of E. histolytica were found in these inhabitants, provided that the isolated zymodemes I, III, IV, VIII, IX, X, XVII and XVIII are recognized as nonpathogenic strains of E. histolytica. Furthermore, the prevalence of intestinal helminths and other protozoan infections showed that these individuals are infected by other agents responsible for diarrhoeal diseases.

Entamoeba histolytica: detection of coproantigens by purified antibody in the capture sandwich ELISA

A sensitive and specific Capture Sandwich ELISA (CSE) was developed using polyclonal purified rabbit antibodies against three different axenic strains of Entamoeba histolytica: CSP from Brazil and HM1 - IMSS from Mexico, for the detection of coproantigens in fecal samples. Immunoglobulin G (IgG) againstis E. histolytica was isolated from rabbits immunized with throphozoites whole extract in two stages: affinity chromatography in a column containing E. histolytica antigens bound to Sepharose 4B was followed by another chromatography in Sepharose antibodies 4B-Protein A. A Capture Sandwich ELISA using purified antibodies was able to detect 70ng of amebae protein, showing a sensitivity of 93% and specificity of 94%. The combination of microscopic examination and CSE gave a concordance and discordance of 93.25% and 6.75%, respectively. It was concluded that CSE is highly specific for the detection of coproantigens of E. histolytica in feces of infected patients, is quicker to perform, easier and more sensitive than microscopic examination.

Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.

Liver abscess caused by Ascaris lumbricoides: case report

A case is reported of a woman who lived in a rural area with a chronic illness that consisted of weight loss and abdominal pain in the epigastrium and upper right quadrant. The initial diagnosis was a mass in the liver, which was later, demonstrated, both by direct and histological examination, to be an abscess caused by Ascaris lumbricoides. Eggs of Ascaris lumbricoides and abundant Charcot-Leyden Crystals were found.

Rectorragias Como Forma de Apresentação de Amebíase

A amebíase é uma doença infecciosa com diferentes formas de apresentação. Trata-se de um doente de 32 anos, sexo masculino, raça caucasiana, natural e residente em Lisboa, admitido na consulta de Proctologia por rectorragias. O exame objectivo e a avaliação laboratorial não mostraram alterações. Na sigmoidoscopia observaram-se úlceras entre 3 e 5 mm na mucosa rectal e da sigmoideia com friabilidade. A histologia revelou: manutenção da arquitectura, sem depleção das células caliciformes, infiltrado inflamatório intenso, misto com muitos polimorfonucleares neutrófilos e agregados linfóides da lâmina própria, criptite e úlcera com exsudado necroinflamatório, identificando-se Entamoeba histolytica. Medicado com metronidazol com resolução clínica, endoscópica e histológica.

PREVALENCE OF Entamoeba histolytica/Entamoeba dispar IN THE CITY OF CAMPINA GRANDE, IN NORTHEASTERN BRAZIL

There is a clear need to perform epidemiological studies to find the true prevalence of Entamoeba histolytica around the world. The evaluation of this prevalence has been hindered by the existence of two different species which are morphologically identical, but genetically different, namely E. histolytica, which causes amebiasis, and E. dispar, which is non-pathogenic. In Brazil, the E. dispar has been detected in communities in the Southeastern (SE) and Northeastern (NE) regions with poor sanitation. However, individuals infected with E. histolytica have been identified in other regions. There is an absence of reports on the prevalence of these parasites in the state of Paraíba, which also has areas with poor sanitary conditions where a high prevalence of the E. histolytica/E. dispar complex has been detected in children from urban slums. The present study evaluated the prevalence of E. histolytica and E. dispar in 1,195 asymptomatic children between two and 10 years of age, living in a sprawling urban slum in Campina Grande, in the state of Paraíba, in Northeastern Brazil. These children were examined and their feces samples were analyzed microscopically. A total of 553 children tested positive for the E. histolytica/E. dispar complex, and 456 of the positive samples were tested with the E. histolytica II® ELISA kit. All 456 samples were negative for the presence of the adhesin E. histolytica specific antigen. The evidence suggests that in this community E. histolytica is absent and E. dispar is the dominant species.

Intestinal parasitic infections and eosinophilia in an human immunedeficiency virus positive population in Honduras

The occurrence of intestinal parasites, their regional distribution and their relations to eosinophilia were studied in 133 human immunodeficiency virus (HIV) positive individuals from Honduras. After signing an informed consent, participants answered a socio-demographic and risk factor questionnaire, a complete physical examination, medical history, and a series of laboratory tests. All participants were HIV positive but not acquired immunodeficiency syndrome positive. Of them, 67% were co-infected with pathogen and non pathogen parasites. Overall occurrence of nematodes was: 44.3% for Trichuris trichiura, 24% for Ascaris lumbricoides, 12% for Hookworm and 7.5% for Strongyloides stercoralis. No cases of Giardia lamblia, acute amebiasis or cryptosporidiasis were diagnosed. Mean eosinophil percents for participants were consistently and significantly higher in infected than in non infected individuals: 22% for Hookworm vs 7.2% (p < 0.001), 11% for Trichuris compared to 5.2% (p < 0.001), 13.2% compared to 7.5% for S. stercoralis (p < 0.05), and 12% compared to 6% for Ascaris cases (p < 0.05). Helminths and non pathogenic protozoa, as single or mixed infections, occurred among the participants. There was a strong correlation between eosinophilia and helminthiasis infections; however, none was identified between CD4 levels and eosinophilia. Because parasitic infections aggravate malnutrition and promote a disbalanced Th2 response in a potentially immuno-compromised host, their effect on HIV disease progression needs further study, mainly in countries were HIV and parasitic infections are highly prevalent.

Systemic acanthamoebiasis associated with canine distemper in dogs in the semiarid region of Paraíba, Brazil

Infections by free-living amoebae can cause systemic disease in animals and humans. We describe the epidemiological, clinical and pathological aspects of disseminated acanthamoebiasis associated with canine distemper in three dogs of the semiarid region of Paraíba, Northeastern Brazil. Affected dogs developed progressive neurological and respiratory signs that progressed to death within in two to 20 days. Gross lesions were irregular and with yellow-reddish nodules randomly distributed in the lungs, heart, kidneys, spleen, lymph nodes, adrenals, and intestine. One dog had foci of malacia in the parietal cortex and another one in nucleus of brain basis. Histologically, pyogranulomas with areas of necrosis and hemorrhage in all organs affected were observed, associated with myriads of intralesional amoebic trophozoites. All three cases were concomitant canine distemper, that possibly triggered immunosuppression in the dogs. The diagnosis was performed through microscopic findings of infection by free-living amoebae and confirmed Acanthamoeba sp. by immunohistochemistry

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica

It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.

Efeito dos componentes obtidos de qualea multiflora sobre modelo tumoral mamário murino e sua influência no sistema imunológico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diagnóstico de enteroparasitoses numa comunidade rural de Mirante da Serra, Rondônia, Brasil

As enteroparasitoses estão entre as infecções mais comuns no homem, apresentando-se com maior intensidade em países subdesenvolvidos por estarem diretamente relacionadas às condições higiênico-sanitárias. Este estudo tem por objetivo investigar a presença de enteroparasitas causadoras de doenças no homem, em uma comunidade do Movimento dos Trabalhadores Rurais Sem Terra (MST) no estado de Rondônia. No acampamento Pe. Ezequiel, município de Mirante da Serra, RO, foram utilizados os métodos direto e de sedimentação espontânea em água para detecção e identificação das formas parasitárias de protozoários e helmintos em material fecal de residentes do mesmo e o teste de ELISA (Ensaio imunoenzimático) para detecção de coprantígeno específico, anti-GIAP (proteina de aderência inibidora da galactose), de Entamoea histolytica (Tachlab, Blacksburg, VA, USA). Foram examinadas 313 amostras fecais pelos testes coproscópicos e 186 pelo teste de ELISA. A prevalência encontrada foi de 33,5% sendo a E. histolystica, Giárdia Lamblia e os ancilostomídeos os parasitas patogênicos mais frequentes. Não foram encontradas diferenças significativas entre parasitismo e idade, mas entre agente causador da infecção e sexo houve significância entre E. histolytica e sexo feminino. Não foi possível determinar fator (es) de risco relacionado (s) às infecções. Quanto ao resultado do ELISA, foi detectado um número maior de amébiase intestinal por este método do que por coproscopia. Considerando ser o acampamento uma área rural sem infra-estrutura ideal, o baixo parasitismo encontrado foi surpreendente. Todavia, a estrutura alternativa do local somado à organização social do grupo, consciência comunitária e formação básica em saúde, constitui fatores preponderantes para a aprovação e/ou controle das enteroparasitoses.

Estudo epidemiológico da amebíase no Estado do Pará utilizando diferentes metodologias para diagnóstico

A epidemiologia da amebíase está sendo reavaliada desde que a E. histolytica (patogênica) foi considerada espécie distinta de E. dispar (não patogênica). Neste estudo, investigou-se a freqüência da amebíase em uma amostra de residentes do Pará por diferentes técnicas de diagnóstico e avaliou-se a patogenia do parasito. Os participantes (n = 845) forneceram material fecal e destes, 191 foram entrevistados quanto aos sintomas de diarréia, cólicas intestinais, constipação, náuseas e vômito. Foram também analisados 8 exsudatos de pacientes com suspeita de amebíase hepática. As amostras foram observadas sob microscopia de luz e a confirmação de E. histolytica feita a partir da pesquisa de antígenos. Um total de 98 amostras fecais e todos os exsudatos foram semeados em meio Pavlova para isolamento e posterior caracterização bioquímica e molecular (identificação de espécie e genotipagem). Isolados de outras regiões do Brasil foram também genotipados. A positividade obtida foi de 29,35% (248/845) e não houve correlação com a faixa etária. A microscopia revelou baixa sensibilidade (45,26%; 74/334), porém elevada especificidade (87,03%; 260/334) quando comparada ao ELISA. Houve relação significativa (OR 4,4026) entre a presença de sintomas e a positividade no ELISA, sendo a diarréia (58,82%) e a cólica intestinal (58,82%) os sintomas mais relatados. Nenhum exsudato foi positivo no exame a fresco, porém 7 foram positivos no ELISA. Obteve-se 22 isolados de material fecal e a caracterização da HE foi possível em 13, dos quais 7 E. histolytica e 6 E. dispar. O DNA de 22 isolados e dos exsudatos foram testados para identificação molecular de espécie e genotipagem. Do total, 16 cultivos (9 cepas mistas, 4 E. dispar e 3 E. histolytica) e 5 exsudatos (todos E. histolytica) amplificaram na PCR. A genotipagem identificou adicional positividade para E. histolytica em um exsudato e revelou diferentes polimorfismos de comprimento para o locus 1-2 de E. histolytica e E. dispar do Pará e de outras regiões do Brasil e um caso de co-infecção por diferentes genótipos de E. dispar. Nossos resultados revelam que a amebíase invasiva é um importante problema de saúde pública em nossa população e grande variedade de genótipos de E. histolytica contribuem para a doença no Brasil.