Anti-Acanthamoeba castellanii activity of alkaloid-enriched extracts and lycorine from the Amaryllidaceae species

Abstract Free-living amoebae of the genus Acanthamoeba are the causative agents of granulomatous encephalitis and keratitis, severe human infections. Bioactive compounds from plants are recognized as an alternative source for the development of new drugs. The Amaryllidaceae is a botanical family able to synthesize a very specific and consistent group of biologically active isoquinoline-like alkaloids. The alkaloidal fractions from the Brazilian species Hippeastrum canastrense, H. diniz-cruziae, H. puniceum, and Crinum x amabile, along with the alkaloid lycorine, were investigated against Acanthamoeba castellanii. The in vitro assays were performed with distinct concentrations of lycorine and alkaloidal fractions, while the cell viability was evaluated by the MTT method upon MDCK cells. Chlorhexidine 0.02% was used as the positive control. The effect of alkaloid fractions was concentration dependent, and 2000 µg mL-1 of H. canastrense and H. diniz-cruziae provided a 100% inhibition. At concentrations of 250, 500, and 1000 µg mL-1, the H. diniz-cruziae alkaloidal fraction showed the lowest cytotoxic effect (5%-7%) and remarkable anti-amoebic activity, demonstrating values of IC50 285.61 µg mL-1, low cytotoxicity (5%-7%), and selectivity index (7.0). Taken together, the results are indicative of the great potential that the alkaloids from H. diniz-cruziae have as new candidates for anti-amoebicidal compounds

A history of over 40 years of potentially pathogenic free-living amoeba studies in Brazil - a systematic review

Free-living amoeba (FLA) group includes the potentially pathogenic genera Acanthamoeba, Naegleria, Balamuthia, Sappinia, and Vermamoeba, causative agents of human infections (encephalitis, keratitis, and disseminated diseases). In Brazil, the first report on pathogenic FLA was published in the 70s and showed meningoencephalitis caused by Naegleria spp. FLA studies are emerging, but no literature review is available to investigate this trend in Brazil critically. Thus, the present work aims to integrate and discuss these data. Scopus, PubMed, and Web of Science were searched, retrieving studies from 1974 to 2020. The screening process resulted in 178 papers, which were clustered into core and auxiliary classes and sorted into five categories: wet-bench studies, dry-bench studies, clinical reports, environmental identifications, and literature reviews. The papers dating from the last ten years account for 75% (134/178) of the total publications, indicating the FLA topic has gained Brazilian interest. Moreover, 81% (144/178) address Acanthamoeba-related matter, revealing this genus as the most prevalent in all categories. Brazil's Southeast, South, and Midwest geographic regions accounted for 96% (171/178) of the publications studied in the present work. To the best of our knowledge, this review is the pioneer in summarising the FLA research history in Brazil.

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work. .

Estratégia multidisciplinar na prevenção e controle de doenças de manifestação clínica na infância / Multidisciplinary strategy for preventing and controlling diseases which manifest themselves clinically in early childhood

Objetivo: Apresentar os resultados do projeto multidisciplinar que consolidou um programa de manutenção de saúde a partir de procedimentos individuais e coletivos para o diagnóstico e prevenção de doenças na infância. Métodos: As atividades se pautaram na análise das condições de saúde de escolares de 6 a 12 anos, pertencentes à rede municipal de ensino da região rural de Campo Largo e metropolitana de Curitiba, Paraná, Brasil. As doenças foram investigadas por meio de anamnese, exame clínico, análise microbiológica de secreção orofaríngea e de saliva; além de exame parasitológico de fezes. Atividades interativas e educacionais apresentando informações sobre transmissão e diagnóstico de doenças como cárie, febre reumática e parasitoses foram realizadas envolvendo a comunidade estudantil e familiar. Os casos clínicos foram encaminhados para tratamento e acompanhamento. Resultados: Dentre 4 837 crianças analisadas, observou-se que 81% apresentaram alto risco de desenvolver cárie dental considerando a correlação entre concentração de Streptococcus mutans na saliva e epidemiologia da doença. Os exames copro-parasitológicos revelaram índices de positividade de 70% em crianças de área rural e de 55% em escolares de área metropolitana. Cinco crianças foram diagnosticadas como portadoras de febre reumática. Conclusão: A elevada ocorrência da doença cárie e parasitoses, bem como a existência de indivíduos portadores de febre reumática, demonstraram a demanda desta população por ações de promoção de saúde que visem à educação sanitária, a profilaxia e tratamento. Atividades de extensão desta natureza são de grande importância para as comunidades escolares assistidas.

Objective: This work was conducted to present the results of a multidisciplinary project which established a health program through individual and collective procedures for the diagnosis and prevention of childhood illnesses. Methods: The health status was evaluated of children between the ages of six and twelve in the rural district of Campo Largo and schools in metropolitan Curitiba in the state of Paraná, Brazil. Students were submitted to anamnesis and clinical examination. Samples of saliva and oropharyngeal secretions were collected for microbiological analysis. In addition, fecal exams were performed to detect intestinal parasites. Information about transmission, diagnosis and prevention of dental caries, rheumatic fever and parasites was imparted to the school community by way of lectures, flyers and play activities. All clinical cases were referred for treatment and follow-up. Results: Out of the 4 837 children analyzed, 81% were at high risk of acquiring dental caries, based on the correlation between the concentration of Streptococcus mutans in the saliva and the epidemiology of the disease. The parasitological tests showed that 70% of the children from the rural areas and 56% of those from metropolitan areas, were infected. Five children tested positive for rheumatic fever.Conclusion: The high prevalence of caries, parasitic infections and the occurrence of rheumatic fever in children point to the urgency for initiatives to promote health through actions aimed at sanitary education, prophylaxis and treatment. Educational actions of this nature are of majorimportance to the school communities attended to.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water / Produção de anticorpos policlonais anti-Cryptosporidium e padronização da imunofluorescência direta para a detecção de oocistos na água

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20 percent of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

INTRODUÇÃO: A produção de anticorpos policlonais anti-Cryptosporidium e sua utilização na imunofluorescência para determinar a presença de Cryptosporidium em água são descritas no presente trabalho. MÉTODOS: Dois coelhos foram imunizados com antígeno solúvel e particulado provenientes de oocistos purificados de Cryptosporidium. O soro produzido foi preparado para a extração de imunoglobulinas G, que foram purificadas e conjugadas com isotiocianato de fluoresceína (FITC). Lâminas contendo quantidades conhecidas de oocistos foram preparadas para determinar a sensibilidade da técnica. Para testar a especificidade foram preparadas lâminas contendo cistos de Giardia duodenalis. RESULTADOS: O conjugado foi usado com sucesso em amostras de água contaminadas experimentalmente com oocistos de Cryptosporidium, sendo capaz de detectar até cinco oocistos/spots que corresponde a uma contaminação de 250 oocistos/mL. CONCLUSÕES: As três imunizações realizadas nos coelhos foram suficientes para produção de anticorpos contra Cryptosporidium; a reação de imunofluorescência direta padronizada permitiu a detecção de cinco oocistos em 20 por cento das amostras; não houve reação cruzada com cistos de Giardia duodenalis.

Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica / Patogenicidade de Entamoeba dispar em cultivo polixênico e monoxênico comparada a uma cepa virulenta de E. histolytica

Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8 percent of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100 percent in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.

Dois isolados de Entamoeba dispar em cultivo polixênico e culturas clonadas deles obtidas foram submetidos à monoxenização utilizando Crithidia fasciculata como organismo associado. O crescimento em cultivo monoxênico dos isolados e clones, bem como sua capacidade de destruir células VERO (efeito citopático) e de produzir lesões hepáticas em hamster foram comparados a uma cepa virulenta de E. histolytica. Os trofozoítos de E. dispar em cultivo monoxênico apresentaram um crescimento nitidamente menor que o de E. histolytica e foram avirulentos tanto no teste in vivo quanto in vitro. Entretanto, isolados e clones de E. dispar em cultivo polixênico exibiram uma atividade lítica moderada sobre as células VERO (1,5 to 41,8 por cento de destruição) e causaram lesões hepáticas em hamster (29 a 100 por cento em prevalência e 0,86 a 4,00 no grau de lesão) tão extensas quanto aquelas causadas pela E. histolytica. Embora E. dispar não seja associada à doença invasiva no homem, a ocorrência de lesões teciduais significativas, causadas por trofozoítos em condições experimentais, indica que esta espécie pode apresentar potencial patogênico considerável quando em presença de bactérias intestinais.

An attempt at reversebility and increase of the virulence of axemic strains of Entamoeba hitolytica

Neste trabalho procuramos verificar se a interacao "in vitro" com bacterias e fragmentos de figado de hamster normal, modificaria o comportamento patogenico de cepas axenicas de E. histolytica avirulentas (ICB-32 e ICB-RPS); virulentas, porem atenuadas (ICB-CSP e HM1) e de media virulencia (ICB-462). Todas as tentativas de tornar virulentas, restabelecer ou aumentar a virulencia das cepas axenicas de E. histolytica utilizadas fracassaram.