Autophagic cell death induction in a clinical isolate of Acanthamoeba sp. treated with methanolic extracts of Pereskia bleo Kuh

Aims@#This study was aimed to investigate the effects of methanol extracts from various parts of the Pereskia bleo Kuhn plant on Acanthamoeba sp. The antioxidant levels of each extract from different plant parts were measured after the extraction process. These extracts were then exposed to Acanthamoeba sp. to assess dose-response, IC50 values, changes in cell morphology, internal cell activity and apoptosis based on alterations in phospholipids.@*Methodology and results@#The total phenolic content, carotenoid estimation and antioxidant activity of the leaves, flowers and fruits of P. bleo were measured based on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) assay. Its anti-amoebic properties were tested using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay for the IC50 determination. The morphological and biochemical changes in the Acanthamoeba sp. were observed under light and fluorescence microscopy using the acridine orange and propidium iodide double staining (AO/PI). The IC50 values of P. bleo leaves, flowers and fruits methanolic extracts were 5.884%, 0.1646% and 20.69%, respectively. Morphological observation displayed shortened acanthapodia with darkened cytoplasms. AO/PI-stained Acanthamoeba sp. cells appear with orange-fluorescent organelles in their green cytoplasm, indicating autophagic cell deaths. Apoptotic and necrotic Acanthamoeba sp. cells were absent based on Annexin V labelling.@*Conclusion, significance and impact of study: @#This study confirmed that the methanolic crude extracts of P. bleo exhibit high cytotoxic potential towards Acanthamoeba sp. trophozoites by inducing an autophagic mode of cell death.

Cultivo de Acanthamoeba spp. en agua apirógena / Acanthamoeba spp. culture in pyrogen-free water / Cultura de Acanthamoeba spp. em água apirogênica

Resumen El objetivo de este estudio fue evaluar el cultivo de cepas de Acanthamoeba spp. en agua destilada estéril apirógena de uso farmacéutico. Se utilizaron dos cepas de genotipo T4 [un aislamiento de encefalitis granulomatosa amebiana (EGA) y una ambiental] y cepas correspondientes a los genotipos T5 y T15. Los quistes de cada una de las cepas se sembraron en placas de Petri con agar no nutritivo con diferentes soluciones (agua destilada estéril apirógena uso médico para preparaciones inyectables, agua destilada filtrada, medio Page) y combinados con suspensiones de Escherichia coli. Las placas se incubaron a 37 °C y se monitorearon diariamente durante 15 días para la detección de trofozoítos. El crecimiento se evaluó mediante examen microscópico directo. Cada cultivo contó con cuatro repeticiones para cada una de las cepas (n=96). En conclusión, se hallaron diferencias estadísticamente significativas en el crecimiento de las cepas por día. Las cepas T5 y T4 (encefalitis amebiana granulomatosa) desarrollaron mayor cantidad de trofozoítos en el primer día respecto de la cepa T15 (H=16,42; p=0,001). En el agua apirógena con E. coli se obtuvo un crecimiento igual a la solución de Page con E. coli (H=24,64; p=0,0001). No se hallaron diferencias estadísticamente significativas en la cantidad de trofozoítos obtenidos en agua apirógena con E. coli y solución de Page con E. coli en la cepa T4 (EGA) (U=4; p<0,05) pero sí en la cepa T4 ambiental (U=0; p<0,05).

Abstract The objective of this study was to evaluate the culture of strains of Acanthamoeba spp. in sterile apyrogenic distilled water for pharmaceutical use. Two T4 genotype strains [one isolate of granulomatous amebic encephalitis (GAE) and one environmental], a T5 and T15 genotype strains were used. The cysts of each of the strains were seeded in Petri dishes with non-nutritive agar with different solutions (pyrogenic sterile distilled water for medical use for injectable preparations, filtered distilled water, Page medium) and combined with Escherichia coli suspensions. Plates were incubated at 37 °C and monitored daily for 15 days for the detection of trophozoites. Growth was assessed by direct microscopic examination. Each medium culture counted four replicates for each of the strains (n=96). Concluding, statistically significant differences were found in the growth of the strains per day. Strains T5 and T4 (granulomatous amebic encephalitis) developed a greater number of trophozoites on the first day compared to strain T15 (H=16.42; p=0.001). In apyrogenic water with E. coli, a growth equal to Page's solution with E. coli was obtained (H=24.64; p=0.0001). No statistically significant differences were found in the amount of trophozoites obtained in apyrogenic water with E. coli and Page's solution with E. coli in strain T4 (GAE) (U=4; p<0.05), but significant differences were found in the environmental T4 strain (U=0; p<0.05).

Resumo O objetivo deste trabalho foi avaliar o cultivo de cepas Acanthamoeba spp. em água destilada apirogênica estéril para uso farmacêutico. Foram utilizadas duas cepas de genótipo T4 [um isolamento de encefalite granulomatosa amebiana (EGA) e uma ambiental], e cepas correspondentes aos genótipos T5 e T15. Os cistos de cada uma das cepas foram semeados em placas de Petri com ágar não nutritivo com diferentes soluções (água destilada estéril apirogênica para uso médico para preparações injetáveis, água destilada filtrada, meio Page) e combinados com suspensões de Escherichia coli. As placas foram incubadas a 37 °C e monitoradas diariamente durante 15 dias para detecção de trofozoítos. O crescimento foi avaliado através de exame microscópico direto. Cada cultura contou com quatro réplicas para cada uma das cepas (n=96). Em conclusão, diferenças estatisticamente significativas foram encontradas no crescimento das cepas por dia. As cepas T5 e T4 (encefalite amebiana granulomatosa) desenvolveram maior número de trofozoítos no primeiro dia em relação à cepa T15 (H=16,42; p=0,001). Em água apirogênica com E. coli, foi obtido crescimento igual ao da solução de Page com E. coli (H=24,64, p=0,0001). Não foram encontradas diferenças estatisticamente significativas na quantidade de trofozoítos obtidos em água apirogênica com E. coli e solução de Page com E. coli na cepa T4 (EGA) (U=4; p%<0,05), mas sim na cepa T4 ambiental (U=0; p<0,05).

Amebiasis cutánea por ameba de vida libre, heraldo ignorado de encefalitis letal. Reporte de caso / Free-living cutaneous amebiasis, ignored herald of lethal encephalitis. Case report

Introducción: Las enfermedades por amebas de vida libre (AVL) son poco frecuentes pero con letalidad mayor al 90% cuando comprometen el sistema nerviosos central (SNC). El cuadro clínico puede confundirse con enfermedades más prevalentes. Reporte de caso: Agricultor de 32 años procedente de Poroto-Trujillo-La Libertad-Perú, inicia enfermedad con una placa eritematosa centrofacial que se diagnosticó como tuberculosis cutánea (TBC), sin mejoría pese a recibir esquema completo de quimioterapia antituberculosa. Siete meses después presenta encefalitis severa falleciendo a los tres días de su ingreso al hospital. En la biopsia de piel se encontraron trofozoitos de AVL. Se plantea el diagnóstico de dermatitis cutánea por AVL complicada con encefalitis granulomatosa amebiana (EGA). No se han documentado casos similares en su lugar de procedencia. Conclusión: En el norte del Perú, las placas cutáneas en personas expuestas al suelo o agua estancada, deben considerarse señales de alerta ante la probabilidad de infección por AVL.

ABSTRACT Introduction: Diseases caused by free-living amoebas (FLA) are rare but with a lethality greater than 90% when they affect the central nervous system (CNS). The clinical picture can be confused with more prevalent diseases. Case report: A 32-year-old farmer from Poroto-Trujillo-La Libertad-Peru, started the disease with a central facial erythematous plaque that was diagnosed as cutaneous tuberculosis (CTB), without improvement despite receiving a complete regimen of anti-tuberculous chemotherapy. Seven months later, he presented severe encephalitis and died three days after his admission to the hospital. FLA trophozoites were found in the skin biopsy. A diagnosis of cutaneous dermatitis due to FLA complicated with granulomatous amebic encephalitis (GAE) is proposed. Similar cases have not been documented in his place of origin. Conclusion: In northern Peru, skin plaques in people exposed to the ground or stagnant water should be considered warning signs of the probability of AVL infection.

Elliptical deep anterior lamellar keratoplasty in severe Acanthamoeba keratitis

Acanthamoeba keratitis (AK) is an intractable infection of the cornea. Penetrating keratoplasty is widely used for the management of severe AK but suffers from complications like graft rejection, endophthalmitis, and glaucoma. Herein, we aimed to describe the surgical technique and the results of elliptical deep anterior lamellar keratoplasty (eDALK) for the management of severe AK. In this retrospective case series, records of consecutive patients with AK poorly responsive to medical treatment who underwent eDALK from January 2012 to May 2020 were reviewed. The largest diameter of infiltration was ?8 mm and did not involve the endothelium. The recipient bed was made by an elliptical trephine, and big bubble or wet-peeling technique was performed. Postoperative best spectacle-corrected visual acuity, endothelial cell density, corneal topographic data, and complications were evaluated. Thirteen eyes of thirteen patients (eight men and five women, 45.54 ± 11.78 years old) were included in this study. The mean follow-up interval was 21.31 ± 19.59 months (range, 12–82 months). At the last follow-up, the mean best spectacle-corrected visual acuity was 0.35 ± 0.27 logarithm of the minimum angle of resolution. The mean refractive and topographic astigmatism were ? 3.21 ± 1.77 and 3.08 ± 1.14 D, respectively. Intraoperative perforation was encountered in one case and double anterior chambers occurred in two cases. One graft developed stromal rejection and one eye developed amoebic recurrence. eDALK can serve as the first-line surgical management of severe AK poorly responsive to medical treatment.

Alternativa rápida de extracción de ADN en quistes de Acanthamoeba para uso en el laboratorio de análisis clínicos / Rapid DNA extraction alternative in cysts of Acanthamoeba for use in clinical analysis laboratory

Resumen El objetivo del estudio fue comparar la extracción de ADN de quistes de Acanthamoeba sp. con un método disponible comercialmente y cuatro no comerciales utilizando tratamiento térmico y ultrasonido para la amplificación por una reacción en cadena de la polimerasa (PCR) convencional, reduciendo tiempos de preparación y extracción de las muestras, como una herramienta para el diagnóstico en el laboratorio clínico. Se utilizó una cepa de Acanthamoeba, genotipo T4, cultivada en agar no nutritivo. Los quistes para analizar, en tres períodos de enquistamiento, se almacenaron a temperatura ambiente. Se extrajo ADN mediante cinco métodos: pretratamiento térmico, ultrasonido y combinaciones de ellos. La PCR se llevó a cabo utilizando cebadores específicos JDP1/JDP2. La concentración y pureza del ADN extraído con los protocolos evaluados revelaron diferencias estadísticamente significativas (p<0,0001). El método E (comercial), el A (térmico) y el B (ultrasonido) lograron los mejores rendimientos en la amplificación del fragmento específico de Acanthamoeba sp. por la PCR convencional.

Abstract The objective of the study was to compare the DNA extraction of Acanthamoeba sp. cysts with a commercially available method and four non-commercial ones, with heat and ultrasound treatment that allows amplification by conventional polymerase chain reaction (PCR), reducing sample preparation and extraction times, such as a tool for diagnosis in the clinical laboratory. To this aim, a strain of Acanthamoeba T4 grown on non-nutrient agar was used. Plates with cysts at three different encystation times were stored at room temperature until the study was carried out. DNA was extracted with five methods that included pretreatments (thermal and ultrasound) or combinations of them. PCR was performed using specific primers JDP1/JDP2. Concentration and purity of DNA revealed statistically significant differences (p<0.0001) between methods. Method E (commercial), method A (thermal) and B (ultrasound) got the best yields in amplifying the specific fragment of Acanthamoeba sp. by conventional PCR.

Resumo O objetivo do estudo foi comparar a extração de DNA de cistos de Acanthamoeba sp. com um método comercialmente disponível e quatro não comerciais utilizando tratamento térmico e ultrassom para amplificação por reação em cadeia da polimerase (PCR) convencional, reduzindo os tempos de preparo e extração das amostras, como ferramenta para o diagnóstico no laboratório clínico. Foi utilizada uma cepa de Acanthamoeba, genótipo T4, cultivada em ágar não nutritivo. Os cistos para analisar foram armazenados em temperatura ambiente, correspondendo a três períodos de encistamento. O DNA foi extraído por cinco métodos: pré-tratamento térmico, ultrassom e combinações deles. A PCR foi realizada usando iniciadores específicos JDP1/JDP2. A concentração e a pureza do DNA extraído com os protocolos avaliados revelaram diferenças estatisticamente significativas (p<0,0001). Os métodos E (comercial), A (térmico) e B (ultrassom) alcançaram os melhores rendimentos na amplificação do fragmento específico de Acanthamoeba sp. por PCR convencional.

Isolamento de amebas de vida livre potencialmente patogênicas em piscinas de uso coletivo localizadas no município de Redenção, Pará, Brasil / Isolation of potentially pathogenic free-living amoebae in swimming pools for collective use located in the municipality of Redenção, Pará, Brazil

Objetivo Isolar amebas dos gêneros Acanthamoeba e Naegleria em piscinas de uso coletivo do município de Redenção, Pará, Brasil. Métodos A identificação dessas amebas se deu a partir de amostras retiradas de piscinas de uso coletivo de quatro clubes da cidade. As análises foram realizadas a partir do exame direto a fresco, cultura, análise morfológica após coloração de Gram e teste de exflagelação, seguindo o protocolo descrito por Carlesso, Artuso e Rott. Resultados As amebas de vida livre foram encontradas em 41,67% das amostras, com predomínio das do gênero Acanthamoeba (63,2%) no exame direto a fresco. No clube A, localizado no setor Serrinha, houve predominância dessas amebas em relação às demais (8,34%). As amostras obtidas por swabs apresentaram 73,69% de formas evolutivas em comparação à análise feita apenas na água das piscinas. A confirmação dos gêneros das amebas presentes nas amostras feita através da coloração de Gram encontrou um perfil de identificação diferente, sendo que nesse exame a ameba de gênero Naegleriaspp. se sobressaiu perante a de gênero Acanthamoeba (61,11% e 38,89%, respectivamente). Conclusão Os resultados do estudo confirmam a presença de isolados potencialmente patogênicos de amebas de vida livre em piscinas de uso coletivo, o que pode significar um risco à saúde pública.

Objective To isolate amoebae of the genera Acanthamoeba and Naegleria in swimming pools for collective use in the municipality of Redenção, Pará, Brazil. Methods The identification of these amoebae was determined from samples of swimming pools for collective use in four private clubs in the city. The analysis methodology was performed based on direct fresh examination, culture, morphological analysis after Gram staining and exflagellation test, following the protocol described by Carlesso, Artuso and Rott. Results The frequency of free-living amoebae was 41.67%, with a predominance of the Acanthamoeba genus (63.2%) in direct fresh examination. At club A, located in the Serrinha sector, the frequency of these amoebae was higher than the others (8.34%). The samples obtained by swabs showed 73.69% of evolutionary forms in comparison to the analysis only of the water in the pools. The confirmation of the genera by Gram stain showed a different identification profile, in which Naegleria spp. stood out before the genus Acanthamoeba (61.11% and 38.89%, respectively). Conclusion In conclusion, the results of the study confirm the presence of potentially pathogenic isolates from free-living amoebae in swimming pools for collective use that may present risks to public health.

Central nervous system infections caused by pathogenic free-living amoebae: An Indian perspective

@#Pathogenic free-living amoebae (FLA), namely Acanthamoeba sp., Naegleria fowleri and Balamuthia mandrillaris are distributed worldwide. These neurotropic amoebae can cause fatal central nervous system (CNS) infections in humans. This review deals with the demographic characteristics, symptoms, diagnosis, and treatment outcomes of patients with CNS infections caused by FLA documented in India. There have been 42, 25, and 4 case reports of Acanthamoeba granulomatous amoebic encephalitis (GAE), N. fowleri primary amoebic meningoencephalitis (PAM), and B. mandrillaris meningoencephalitis (BAE), respectively. Overall, 17% of Acanthamoeba GAE patients and one of the four BAE patients had some form of immunosuppression, and more than half of the N. fowleri PAM cases had history of exposure to freshwater. Acanthamoeba GAE, PAM, and BAE were most commonly seen in males. Fever, headache, vomiting, seizures, and altered sensorium appear to be common symptoms in these patients. Some patients showed multiple lesions with edema, exudates or hydrocephalus in their brain CT/MRI. The cerebrospinal fluid (CSF) of these patients showed elevated protein and WBC levels. Direct microscopy of CSF was positive for amoebic trophozoites in 69% of Acanthamoeba GAE and 96% of PAM patients. One-fourth of the Acanthamoeba GAE and all the BAE patients were diagnosed only by histopathology following autopsy/biopsy samples. Twenty-one Acanthamoeba GAE survivors were treated with cotrimoxazole, rifampicin, and ketoconazole/amphotericin B, and all eleven PAM survivors were treated with amphotericin B alongside other drugs. A thorough search for these organisms in CNS samples is necessary to develop optimum treatment strategies.

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.

Three Decades of Contact Lens-associated Microbial Keratitis in a Referral Hospital in São Paulo, Brazil / Ceratite Microbiana Associada a Lentes de contato em um Hospital de Referência em São Paulo, Brasil

ABSTRACT Purpose: The aim of this study was to analyze patient data and the laboratory results of corneal samples collected from patients followed at the Ophthalmology Department, Hospital São Paulo, Brazil over a 30-year period, and correlate the analize with contact lens wearing. Methods: Corneal samples from patients diagnosed clinically with microbial keratitis between January 1987 and December 2016 were included in this study. Cultures that were positive for bacteria, fungi, and Acanthamoeba spp. were analyzed retrospectively. To ascertain if the number of patients with contact-lens-associated microbial keratitis (as a risk factor for microbial infection) changed over time, the analysis was divided into three decades: 1987-1996, 1997-2006, and 2007-2016. Information pertaining to patient gender and age, as well as type of organism isolated, were compared among these periods. Results: The corneal samples of 10.562 patients with a clinical diagnosis of microbial keratitis were included in the study, among which 1.848 cases were related to the use of contact lenses. The results revealed that the frequency of contact-lens-associated microbial keratitis increased over the last two decades. Overall, females had contact-lens-associated microbial keratitis more frequently (59.5%). Patients aged 19-40 years also had contact-lens-associated microbial keratitis more frequently in all study periods. Staphylococcus spp. and Pseudomonas spp. were the most frequent Gram-positive and Gram-negative bacteria, respectively, in the microbial keratitis groups. Among the fungal cases of microbial keratitis, filamentous fungi were the most frequent fungi over the entire study period, with Fusarium spp. being the most frequent fungi in the group with microbial keratitis not associated with contact lens wearing (p<0.001). Samples positive for Acanthamoeba spp. and Pseudomonas spp. were significantly correlated with contact-lens-associated microbial keratitis (p<0.001). Conclusions: Females and young adults aged 19-40 years exhibited the highest frequency of contact-lens-associated microbial keratitis in our study. Staphylococcus spp. and Fusarium spp. were the predominant bacteria and fungi, respectively, isolated from corneal samples. Pseudomonas spp. and Acanthamoeba spp. were significantly correlated with contact-lens-associated microbial keratitis in this study.

RESUMO Objetivo: O objetivo deste estudo foi analisar da dos epidemiológicos de pacientes e resultados laboratoriais para todas as amostras de córnea coletadas de pacientes atendidos no Departamento de Oftalmologia do Hospital São Paulo, Brasil, durante um período de 30 anos e correlacionar com o uso de lentes de contato. Métodos: Amostras de córnea de pacientes com diagnóstico clínico de ceratite microbiana (de janeiro de 1987 a dezembro de 2016) foram incluídas neste estudo. Resultados laboratoriais para culturas positivas para bactérias, fungos e Acanthamoeba spp. foram analisados retrospectivamente. Para verificar se o número de pacientes com ceratite microbiana associada à lente de contato, fator de risco para infecção microbiana, mudou ao longo do tempo, a análise foi dividida em três décadas: 1987-1996, 1997-2006 e 2007-2016. As informações incluindo o sexo do paciente, idade e tipo de organismo isolado foram comparadas entre os períodos. A análise estatística foi realizada no software SAS/STAT 9.3 e SPSS (v20.0). Resultados: Amostras de córnea de 10.562 pacientes com ceratite microbiana foram incluídas no estudo, das quais 1.848 foram relacionadas ao uso de lentes de contato. Os resultados revelaram que a frequência de ceratite microbiana associada à lente de contato aumentou nas últimas duas décadas analisadas. No geral, os homens compreendiam uma proporção maior do grupo ceratite microbiana não associada à lente de contato (CMNLC) (60,3%) e as mulheres eram mais frequentes no grupo ceratite microbiana associada à lente de contato (59,5%). Pacientes com idade entre 19 e 40 anos foram mais frequentemente observados no grupo ceratite microbiana associada à lente de contato em todos os períodos. Staphylococcus spp. foi a bactéria Gram-positiva mais frequentes, enquanto Pseudomonas spp. foi a bactéria Gram-negativa nos grupos ceratite microbiana. Entre os fungos ceratite microbiana, os fungos filamentosos foram os fungos mais frequentes durante todo o período do estudo, com Fusarium spp. sendo o mais frequente no grupo ceratite microbiana não associada à lente de contato. Acanthamoeba spp. e Pseudomonas spp. amostras positivas foram significativamente correlacionadas com ceratite microbiana associada à lente de contato. Conclusões: A maior prevalência de ceratite microbiana associada à lente de contato no nosso estudo foi observada em mulheres e adultos jovens com idade entre 19 e 40 anos. Staphylococcus spp. e Fusarium spp. foram as bactérias e fungos predominantes isolados nas amostras da córnea. Pseudomonas spp. e Acanthamoeba spp. foram significativamente correlacionados a ceratite microbiana associada à lente de contato neste estudo.

Encefalitis por ameba de vida libre: reporte de caso / Free-living amoeba encephalitis: case report

La encefalitis amebiana granulomatosa es una enfermedad considerada atípica debido a la baja incidencia desde que fue descubierta. Sin embargo, conforme han avanzando los métodos diagnósticos, en retrospectiva, se ha descrito la presencia de esta enfermedad desde principios del siglo XX, identificando a Acanthamoeba spp y Balamuthia mandrillaris como agentes más comunes.Es una enfermedad de difícil diagnóstico debido al amplio abanico clínico que puede presentar, siendo el sitio anatómico dentro del sistema nervioso central quien determinará estas características. El conocimiento y sospecha de la misma permitirá un abordaje terapéutico oportuno aunque, en la mayoría de los casos, con un desenlace fatal para el paciente.A continuación se presenta el caso clínico de un paciente masculino de 26 años de edad originario de la ciudad de Durango, México, sin antecedentes de importancia, de ocupación ladrillero, quien sufre golpe con ladrillo en región naso-orbitaria, ingresando al hospital al tercer día del evento con diagnóstico de celulitis periorbitaria izquierda y nasal, cefalea de leve intensidad, rinorrea abundante, hemiparesia fasciocorporal izquierda, parestesia de extremidad superior izquierda, diplopía y vértigo postural. Los estudios imagenológicos revelaron lesiones heterogéneas en el lóbulo occipital, hemisferio cerebeloso izquierdo y tallo cerebral. Se realizó escisión de lesión tumoral cerebelar, con estudio anatomopatológico, reportando encefalitis granulomatosa por presencia de ameba de vida libre. El paciente evolucionó hacia deterioro rostro-caudal declarándose su fallecimiento a los 16 días de su estancia intrahospitalaria.En el Estado de Durango, México, no se cuenta con casos reportados por infección de ameba de vida libre

Granulomatous amoebic encephalitis is a disease considered atypical due to the low incidence since it was discovered. However, as diagnostic methods have progressed, in retrospect, the presence of this disease has been described since the beginning of the 20th century, identifying Acanthamoeba spp. and Balamuthia mandrillaris as the most common agents.A disease that is difficult to diagnose due to the wide clinical range that it can present, being the anatomical site within the central nervous system that will determine these characteristics. The knowledge and suspicion of it will allow a timely therapeutic approach although, in most cases, with a fatal outcome for the patient.The clinical case is presented below of a 26 year old male patient from the city of Durang, México, with no significant history, of brick occupation, who suffers a blow with a brick in the naso-orbital region, follows, entering our hospital on the third day of the event with a diagnosis of left periorbital and nasal cellulitis, mild headache, profuse rhinorrhea, left fasciocorporeal hemiparesis, left upper limb parestesia, diplopia, and postural vertigo.Imaging studies were performed observing heterogeneous lesions in the occipital lobe, left cerebellar hemisphere and brain stem; excision of the cerebellar tumor lesion being carried out, with pathological study, reporting granulomatous encephalitis due to the presence of free-living amoeba. The patient progresses to face-caudal deterioration, declaring his death after 16 days of hospital stay.Within the state of Durango, Mexico, there are no reported cases of free-living amoeba infection

Imagenología diferencial por microscopia confocal in vivo entre la epiteliopatía por acanthamoeba y la queratitis por herpes simple / In vivo confocal microscopy imaging for the differential diagnosis of Acanthamoeba epitheliopathy and herpes simplex keratitis

La queratitis por Acanthamoeba es una infección corneal de baja incidencia, condicionada por varios factores, pero con manifestación clínica variada y sintomatología típica. En su fase epitelial debe ser diferenciada de otras queratitis, específicamente de la queratitis por herpes simple, por la similitud entre ambas en cuanto a las características de la lesión corneal. La microscopia confocal in vivo constituye una alternativa diagnóstica. Es una biopsia fotográfica en cuyas imágenes podemos describir los quistes y trofozoítos de Acanthamoeba desde etapas iniciales, que nos ayudan a diferenciarla de otros tipos de queratitis e iniciar el tratamiento precoz. Se realizó una búsqueda de artículos publicados, con el objetivo de mostrar las imágenes por microscopia confocal de la fase epitelial de la infección corneal por Acanthamoeba y herpes simple. Se utilizó la plataforma Infomed, específicamente la Biblioteca Virtual de Salud(AU)

Acanthamoeba keratitis is a low-incidence corneal infection caused by several factors and characterized by a variety of clinical manifestations and typical symptoms. In its epithelial phase, it should be differentiated from other keratitis, particularly from herpes simplex keratitis, due to the similar characteristics of the corneal lesion. In vivo confocal microscopy is a diagnostic alternative consisting in a photographic biopsy showing images of Acanthamoeba cysts and trophozoites since their initial stages, thus allowing differentiation from other types of keratitis and the initiation of early treatment. A search was conducted of published papers with the purpose of showing confocal microscopy images of the epithelial phase of Acanthamoeba and herpes simplex corneal infection. Use was made of the platform Infomed, specifically the Virtual Health Library(AU)

Presencia de Acanthamoeba spp. en piscinas recreativas al aire libre de la ciudad de Córdoba, Argentina / Presence of Acanthamoeba spp. in outdoor recreational swimming pools in Córdoba city, Argentina / Presença de Acanthamoeba spp. em piscinas recreativas ao ar livre da cidade de Córdoba, Argentina

Resumen El objetivo del estudio fue determinar la presencia de Acanthamoeba spp. en piscinas de uso recreacional, al aire libre, de la ciudad de Córdoba, Argentina. Se recolectaron 30 muestras de agua correspondientes a un total de 10 piscinas. Estas se sembraron en agar no nutritivo en presencia de Escherichia coli en solución de Page. Luego de 72 horas de incubación a 37 °C, la identificación genérica se realizó mediante criterios morfológicos. La prueba de transformación amebo-flagelar se realizó para diferenciar amebas de vida libre que desarrollan trofozoítos flagelados, como Naegleria. Se midieron parámetros fisicoquímicos en cada una de las piscinas. Se identificó Acanthamoeba spp. en el 20% del total de las muestras. Las piscinas en las que se aisló este parásito presentaron niveles no detectables de cloro residual, pH moderadamente alcalino y temperatura templada. Este estudio demuestra la presencia de Acanthamoeba spp. en piscinas recreativas de Córdoba, lo que puede representar un potencial riesgo para la salud pública.

Abstract The aim of the study was to determine the presence of Acanthamoeba spp. in outdoor recreational pools, in Córdoba city, Argentina. Thirty water samples corresponding to a total of 10 pools were collected. These samples were sown on a non-nutritive agar in the presence of Escherichia coli in Pages's solution. After 72 hours of incubation at 37 °C, the generic identification was done based on morphological criteria. The amoebo-flagellate transformation test was performed to differentiate from genera that develop flagellated trophozoites, such as Naegleria. Physicochemical parameters were measured in each of the pools. Acanthamoeba spp. was identified in 20% of the samples. The pools, where this parasite was isolated, presented undetectable levels of residual chlorine, moderately alkaline pH and warm temperature. This study demonstrates the presence of Acanthamoeba spp. in recreational pools in Córdoba, which may represent a potential risk to public health.

Resumo O objetivo do estudo foi determinar a presença de Acanthamoeba spp. em piscinas para uso recreativo, ao ar livre, na cidade de Córdoba, Argentina. Foram coletadas 30 amostras de água correspondentes a um total de 10 piscinas. Elas foram semeadas em ágar não nutritivo na presença de Escherichia coli em solução de Page. Após 72 horas de incubação a 37 °C, a identificação genérica foi realizada utilizando critérios morfológicos. O teste de transformação amebo-flagelar foi realizado para diferenciar amebas de vida livre que desenvolvem trofozoítos flagelados, como Naegleria. Parâmetros físico-químicos foram medidos em cada uma das piscinas. Acanthamoeba spp. foi identificada em 20% do total das amostras. As piscinas onde este parasita foi isolado apresentaram níveis indetectáveis de cloro residual, pH moderadamente alcalino e temperatura temperada. Esse estudo demonstra a presença de Acanthamoeba spp. em piscinas recreativas de Córdoba, o que pode representar um risco potencial para a saúde pública.

Risk factors and treatment outcome of recurrent acanthamoeba keratitis after corneal transplantation / 中华实验眼科杂志

Objective:To investigate the risk factors and treatment outcome of recurrent Acanthamoeba keratitis (AK) after corneal transplantation. Methods:A serial case-observational study was carried out.Twenty-eight eyes of 28 patients with AK who underwent corneal transplantation in Shandong Eye Hospital from January 2012 to January 2019 were enrolled.All the eyes received corneal transplantation from failing to respond to topical and systemic anti- Acanthamoeba medical therapy, including 13 eyes that received penetrating keratoplasty (PKP) and 15 eyes that received lamellar keratoplasty (LKP). The corneal lesion was removed by a trephine with a diameter of 0.5 mm over infiltration area during PKP or LKP.The clinical features of recurrent AK were summarized, including recurrence time, site and signs, and the risk factors of AK recurrence were analyzed.Local and systemic anti- Acanthamoeba medical therapy was performed in all relapsed eyes, and secondary surgery was performed for the eyes with poor response to medication.The therapeutic outcome of recurrent AK was evaluated.The study adhered to the Declaration of Helsinki.This study protocol was approved by an Ethics Committee of Shandong Eye Hospital (No.201112). Results:In the 28 eyes, 7 eyes (25%) appeared recurrent AK after keratoplasty, including 2 eyes after PKP and 5 eyes after LKP.There was no significant difference in the recurrence rate between the two methods ( P=0.396). The recurrence rate of eyes that had used glucocorticoids drugs before operation was 57.14% (4/7), which was significantly higher in comparison with 14.29% (3/21) of eyes without glucocorticoids before surgery ( P=0.043). The recurrence rate of eyes with ulcer diameter ≥8.2 mm was 50.00% (5/10), which was significantly higher than 11.11% (2/18) of eyes with ulcer <8.2 mm ( P=0.036). The recurrent lesions began at the edge of implant bed accounted for 85.71% (6/7), and the recurrent lesions located below graft accounted for 14.29% (1/7). In 7 eyes with recurrent AK, 6 eyes were completely cured.Among recurrent AK eyes after LKP, 2 eyes were cured by long-term medical therapy, and 2 eyes were cured by extended-diameter LKP, and another 1 eye was cured by conjunctival flap covering surgery.One eye with recurrent AK after PKP was cured by extended-diameter PKP. Conclusions:The risk factors of recurrent AK after surgery are application of glucocorticoids before surgery and big lesions.Recurrent AK after surgery is curable by individualized therapy targeting to different clinical characteristics.

First report of detection of IgA anti-Acanthamoeba antibodies among Saudi population and amoeba isolation from their surroundings

@#Acanthamoeba is an opportunistic protozoan pathogen which is found in diverse environment worldwide. Being ubiquitous nature of this amoeba we come across it in our daily life. Acanthamoeba species are recognized as human pathogens; that may cause blinding keratitis and rare but fatal granulomatous encephalitis involving central nervous system. To date, there is not a single report in literature demonstrating anti-Acanthamoeba antibodies among the Saudi population, and thus aim of the present study. Using ELISA, we identified the antibody level in the local population. Our results represent the secretory IgA antiAcanthamoeba in mucosal secretions from 133 individuals aged 15–60 years. The antiAcanthamoeba antibody prevalence rate was > 80%, and no considerable differences were observed between prevalence in males (80.28%) and that in females (80.64%). In addition, environmental sources (soil and water) from the environment of the participants in our study were evaluated for amoeba incidence. The amoeba was identified by morphological characteristics of cysts or trophozoites on non-nutrient agar plates grown with E. coli. Overall, 58.75% of samples from water and 32.85% of those from soil were culture positive for outgrowth of amoeba on non-nutrient agar plates. Furthermore, PCR was carried out with genus-specific primers to confirm the presence of Acanthamoeba DNA. Our results revealed that about 68% of cultures from water and 43% of those from soil were successfully amplified and proved to be amoeba DNA. Interestingly, a few samples yielded more than one product, which suggests that some other amoebic species may be present in the same sample (MAC-W1 and MADW1). To the best of our knowledge, we described for the first time the amoeba isolation from the participant’s close environment and antibodies level among Saudi population. Our future studies will be focused on additional molecular characterization of isolated amoeba and their pathogenic potential which could be a possible threat for the community.

Value of fluorescence staining in histopathological diagnosis of Acanthamoeba keratitis / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the application value of fluorescent staining technique in the detection of amoebic pathogens in corneal tissue biopsy, and to apply fluorescent staining technique in the histopathological diagnosis of Acanthamoeba keratitis(AK), comparing the results with those of hemotoxyiln-eosin staining(HE staining)and periodic acid-schiff staining(PAS staining), and analyzing the sensitivity and specificity of these three staining methods.<p>METHODS:Specimens of infected corneal tissue were collected from 74 cases(75 eyes), and then they were divided into an AK group and a non-Acanthamoeba keratitis(NAK)group based on the results of corneal scraping, culture and histopathological diagnosis. The tissues of consecutive sections were stained with HE staining, PAS staining and fluorescence respectively, and the sensitivity and specificity of the three staining methods for the diagnosis of AK were analyzed. Area under the curve(AUC)was calculated using the receiver operating characteristic(ROC)curve. Further analysis was performed to count the number of Acanthamoeba pathogens found by the three staining methods under the same magnification field of view at the same site, and to clarify the diagnostic value of fluorescent staining technique for AK.<p>RESULTS: The sensitivity of HE staining was 69%(27/39)with a specificity of 92%; the sensitivity of PAS staining was 62%(24/39)with a specificity of 97%, and the sensitivity of fluorescent staining was 95%(37/39)with a specificity of 97%. There were differences in the sensitivity of the three staining methods for the diagnosis of AK(χ2=19.857, <i>P</i><0.001), and pairwise comparison revealed that the differences between HE staining and fluorescent staining, PAS staining and fluorescent staining for the diagnosis of AK were statistically significant(<i>P</i>=0.003,<0.001), while the difference in sensitivity between HE staining and PAS staining for the diagnosis of AK was not statistically significant(<i>P</i>=0.978). The maximum AUC was 0.960 for fluorescence staining, followed by 0.804 for HE staining and 0.794 for PAS staining, respectively. The median number of amoeba cysts detected by HE staining, PAS staining and fluorescent staining at the same site under the same magnification field of view was 4(0, 11), 2(0, 9)and 12(3, 33), respectively(χ2=56.561, <i>P</i><0.001). Pairwise comparison revealed that the differences in the number of amoeba cysts found by HE staining and fluorescence staining, PAS staining and fluorescence staining were statistically significant(<i>P</i><0.001), while the difference in the number of amoeba cysts found by HE staining and PAS staining was not statistically significant(<i>P</i>=0.210). Fluorescently stained histopathological sections make it easier to identify amoebic pathogens.<p>CONCLUSION:Fluorescent staining technique is more sensitive to histopathological diagnosis of AK than HE staining and PAS staining, which can significantly improve the positive rate of detection of amoebic pathogens.

Anti-acanthamoebic activity of methanolic extract of Piper sarmentosum leaves

Aims@#Piper sarmentosum or locally known as Kaduk, is a tropical herb plant that was investigated for its phenolic content by previous researchers. The present study aimed at the analysis of crude methanolic extract of P. sarmentosum leaves for phenolic compounds identification and its anti-amoebic properties against pathogenic Acanthamoeba castellanii.@*Methodology and results@#Folin-Ciocalteu assay was used to determine P. sarmentosum leaves methanolic extract (PSLME)’s total phenolic content (TPC). The extract was further characterized by using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses to determine the chemical constituents in methanolic PSLME extract. The cytotoxicity of the extract was evaluated through the determination of inhibition concentration for half of cell population (IC50) of pathogenic A. castellanii followed by cell morphological analysis using inverted light and scanning electron microscopies. Acridine-orange/Propidium iodide (AOPI) staining was also conducted to determine the integrity of cell membrane for quantitative analysis. The results demonstrated that the TPC from PSLME was 142.72 mg [GAE]/g with a total of 33 phenolic compounds identified. The IC50 value obtained for A. castellanii was low (74.64 μg/mL) which indicates promising anti-acanthamoebic activity. Microscopy analyses showed that the plant extract caused cells encystment, in which exhibited by distinctive morphological changes on the cells shape and organelle, as well as shortening of acanthopodia. The dual staining and its quantitative analysis prove compromised membrane integrity in the treated amoeba.@*Conclusion, significance and impact of study@#This finding provides the evidence that PSLME contains active phenolic compounds contributing to the anti-acanthamoebic activity on pathogenic Acanthamoeba species.

Encefalitis letal por amebas de vida libre en tres miembros de una familia, Tumbes, Perú / Lethal encephalitis due to free-living amoebae in three members of a family, Tumbes, Peru

Las amebas de vida libre (AVL) son parásitos protozoarios que ocasionalmente causan enfermedad en humanos luego del contacto con alguna fuente de aguas o suelo contaminado. Se describe una serie de tres casos de encefalitis con desenlace fatal en un grupo familiar, que tuvo lugar en el departamento de Tumbes entre diciembre de 2019 y febrero de 2020. En la muestra de líquido cefalorraquídeo del primer caso se identificó Acanthamoeba sp. Los tres casos presentaron como antecedente haber entrado a una piscina meses antes de la enfermedad. En Tumbes no hay registro previo de casos de encefalitis por AVL y no se han reportado a nivel mundial su ocurrencia en un conglomerado familiar.

Free-living amoebas (FLA) are protozoan parasites that occasionally cause disease in humans after contact with contaminated water or soil. We describe a series of three cases of encephalitis in a family group with a fatal outcome that occurred in the Tumbes region between December 2019 and February 2020. Acanthamoeba sp. was identified in the cerebrospinal fluid sample from case 1. All three cases had a history of entering a swimming pool months before the disease. In the Tumbes region there is no previous record of encephalitis due to FLA and its occurrence in a family cluster has never been reported before.

Encefalitis amebiana granulomatosa por Amebas de Vida Libre en un paciente pediátrico / Granulomatous amoebic encephalitis due to free-living amebas in a pediatric patient

Introducción: Las amebas de vida libre (AVL) Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, son protozoos ampliamente distribuidos en la naturaleza. Son microorganismos oportunistas, que afectan preferentemente al sistema nervioso central causando cuadros de meningoencefalitis amebiana primaria o de encefalitis granulomatosa amebiana (EGA), ambas patologías de alta mortalidad. Descripsión del Caso: Paciente femenina de 10 años, que ingresa a nuestro hospital por presentar hemiparesia braquiocrural izquierda, vómitos y cefalea de 72 horas de evolución. Se realiza tomografía computada y Resonancia Magnética en la que se identifica una lesión expansiva, sólido-quística ubicada en la región fronto derecha. Se realiza exéresis completa de la misma y se implementa tratamiento farmacológico lográndose la restitución ad-integrum de la función motora y la resolución de la EGA a 30 meses de seguimiento. Discusión: La encefalitis granulomatosa amebiana es una patología causada por amebas de vida libre. Su presentacíon clínica puede ser indistinguible de otras causas de leptomeningitis o encefalitis, como las de origen bacteriano, viral o por micobacterias lo que impide conocer su real incidencia. Su tratamiento antiparasitario es muy complejo y su evolución es habitualmente fatal. Conclusión: Reportamos un caso de Encefalitis Amebiana Granulomatosa en una paciente pediátrica inmunocompetente, con excelente evolución.

Introduction: Free-living amoebas (FLA) Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, are protist widely distributed in nature. Are opportunistic microorganisms, preferentially affect the central nervous system causing primary amoebic meningoencephalitis or amoebic granulomatous encephalitis (AGE), both with high mortality. Case report: A 10 year-old female patient was admitted with a three-day history of left hemiparesis accompanied with headaches and vomiting. Computed tomography and magnetic resonance were performed, in which an expansive solid cystic mass was observed in the right fronto-parietal region. Complete resection was performed and pharmacological treatment was started, achieving complete restitution of motor function and resolution of AGE after 30 months of follow-up. Discussion: AGE is a desease caused by free-living amoebas. Its clinical presentation is similar to other leptomeningitis or encephalitis of different etiology such as bacterial, viral or by mycobacterial, which cannot know its real incidence. Its pharmacological treatment is complex and its evolution is usually fatal. Conclusion: We report a case of Amoebic Granulomatous Encephalitis in an immunocompetent pediatric patient with good outcome.

Encefalitis por amebas de vida libre: herramientas actuales para el diagnóstico y tratamiento tempranos / Free-living amoeba encephalitis: current tools for early diagnosis and treatment

Introducción: Naegleria fowleri, Acanthamoeba spp. y Balamuthia mandrillaris son amebas de vida libre que producen daños sustanciales del sistema nervioso central y cuyo diagnóstico premortem es poco frecuente. Objetivo: Proveer una visión general de los aspectos clínico-epidemiológicos y las posibilidades diagnósticas más eficaces en la meningoencefalitis amebiana primaria y la encefalitis granulomatosa amebiana, así como las experiencias de tratamiento expuestas en la literatura reciente. Métodos: Se realizó una revisión bibliográfica sobre las amebas de vida libre. Se analizó la información obtenida de artículos científicos en la base de datos Google Scholar®, PubMed y las citas relacionadas por el programa en PubMed Central. Análisis y síntesis de la información: En primer término del análisis de la meningoencefalitis amebiana primaria y la encefalitis granulomatosa amebiana, resalta la diferenciación de sus características clínicas y epidemiológicas, también que el líquido cefalorraquídeo presenta gran relevancia para el diagnóstico de la meningoencefalitis amebiana primaria. Sin embargo, para los casos presuntivos de la encefalitis granulomatosa amebiana el examen del líquido cefalorraquídeo depende de la valoración riesgo-beneficio y se ha obtenido mayor positividad con el diagnóstico histopatológico de biopsias premortem. En general, se acrecienta la implementación de la investigación del ADN por diferentes métodos que brindan certeza de cada una de las especies de AVL causantes de enfermedad neurológica. El aislamiento en cultivo confirmatorio de N. fowleri, Acanthamoeba spp. y B. mandrillaris presenta diferencias en la factibilidad de crecimiento en diversos medios de acuerdo con la especie y limitaciones adicionales. Conclusiones: La mortalidad asociada con las infecciones del sistema nervioso central por amebas de vida libre permanece alta, aunque varios estudios brindan experiencias útiles a partir de los casos de pacientes que han sobrevivido. Resulta importante tener en cuenta que el diagnóstico rápido de la infección es clave para un tratamiento exitoso(AU)

Introduction: Naegleria fowleri, Acanthamoeba spp. and Balamuthia mandrillaris are free-living amoebae of infrequent premortem diagnosis which cause substantial damage to the central nervous system. Objective: To provide an overview of the clinical-epidemiological aspects and the most effective diagnostic possibilities in primary amebic meningoencephalitis and granulomatous amebic encephalitis, as well as their treatment experiences in recent publications.. Methods: A bibliographic review was conducted about free-living amoebae. An analysis was performed of data obtained from scientific papers published in the databases Google Scholar and PubMed, and the citations listed by the PubMed Central program. Data analysis and synthesis: As principal term of the analysis of primary amebic meningoencephalitis and amebic granulomatous encephalitis, the differentiation of their clinical and epidemiological characteristics stands out, also that cerebrospinal fluid is highly relevant for the diagnosis of primary amebic meningoencephalitis. However, for presumptive cases of amoebic granulomatous encephalitis, the examination of cerebrospinal fluid depends on the risk-benefit assessment and greater positivity has been obtained with the histopathological diagnosis of premortem biopsies. In general, the implementation of DNA research by different methods provided accurate information about each one of the free-living amoeba species that cause neurological disease. Confirmatory culture isolation of N. fowleri, Acanthamoeba spp. and B. mandrillaris revealed growth feasibility differences between diverse media depending on the species and additional limitations. Conclusions: Mortality associated to central nervous system infections caused by free-living amoebae remains high. Studies describing cases of patients who have survived constitute useful material. It is important to bear in mind that fast diagnosis of the disease is crucial to treatment success(AU)