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.

Primary amoebic meningoencephalitis in children: a case report and literature review / 中华实用儿科临床杂志

A case of primary amoebic meningoencephalitis (PAM) treated in the First Affiliated Hospital of Xinxiang Medical University in March 6, 2022 was reported.The proband was a 14-year-old boy, who was admitted to the hospital because of " fever, headache and vomiting for 2 days" . Metagenomic next-generation sequencing (mNGS) was consequently conducted to examine the pathogens in cerebrospinal fluid.Naegleria infection was detected, so the child was diagnosed with PAM.The disease developed rapidly, and the patient died 29 hours after admission.In the paper, a total of 13 studies were reviewed, and 15 children with PAM were reported.Of the reported cases, only 1 case survived, 14 cases died.PAM had a low incidence, a dangerous condition, and high mortality.Most cases were diagnosed by autopsy or pathogen diagnosis in cerebrospinal fluid.This case is confirmed by mNGS of pathogens, and it is rarely reported at home and abroad.

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.

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.

Transmission characteristics and prevention measures of Naegleria fowleri： a review / 上海预防医学

Objective To review the Naegleria fowleri and primary amoebic meningoencephalitis caused by the naegleria fowleri， so as to strengthen the public awareness of the disease and its prevention. Methods We reviewed the literatures and reports， and summarized the following information： epidemic status， life cycle and characteristics， environmental distribution and infection routes， pathogenesis and clinical manifestations， diagnosis and treatment， prevention and control measures. Results Primary amoeba meningoencephalitis is a water-borne disease caused by the Naegleria fowleri， which enters human body through the nose and arrives at the central nervous system through olfactory nerve， leading to hemorrhagic and necrotic meningitis. Although the disease is rare， it has a fatality rate of 95%. Most of the patients reported were healthy children or young adults who had nasal contact with water contaminated with Naegleria fowleri a week before symptoms appeared. At present， its pathology can be divided into contact mechanism and non-contact mechanism. In contact mechanism， the Naegleria fowleri relies on the phagocytosis of food cup on its surface and the release of cytolytic molecules that directly destroy host cells. In non-contact mechanism， the Naegleria fowleri has toxic effects by secreting proteins. The clinical symptoms are episodic， with an average time of five days from illness to death. At beginning， the symptoms may include headache， vomiting， fever and other influenza-like symptoms， followed by central nervous system symptoms， such as stiff neck， drowsiness， anorexia， fear and so on. Intravenous or intrathecal injection of amphotericin B is considered to be the basic treatment of this disease， which can be combined with other adjunct therapies such as ventricular shunt and cooling to reduce encephaledema. Conclusion Primary amoebic meningoencephalitis is a rare but highly fatal disease. We should strengthen the public awareness of the disease and its prevention， especially to avoid nasal contact with contaminated water. We should also strengthen scientific research， improve the level of diagnosis， and develop effective drugs to prevent the disease before it happens.

Occurrence of The Pathogenic Amoeba Naegleria fowleri, Pathogenesis, Diagnosis, and Treatment Options

@#Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM) that infects the central nervous system (CNS). The amoeba is present ubiquitously. The infection is rare but has a high mortality rate. The pathogenic amoeba reaches the host through the nasal passage and migrates along the olfactory nerves to reach the human brain and cause severe destruction of the CNS. As Malaysia has a large population that practising ablutions in daily routine, the risk of infection increases. Two mechanisms associated with the infection include contact-dependent and contact-independent. Signs and symptoms vary from early stage to later stage of infection. CSF and brain biopsy are the common specimens collected used to diagnose the PAM infection and usually detect and identify by PCR method. As for treatment, the CDC of United States America has recommended the usage of miltefosine that provides promising in vitro therapy against N. fowleri. This review will discuss more on the occurrence of N. fowleri, pathogenicity, diagnostic tools, and pharmacotherapy approach against N. fowleri infection.

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)

Primary amoebic meningoencephalitis in an infant

Primary amoebic meningoencephalitis is rare but fatal disease encountered in immunocompetent individuals. Here, we present a case of a previously healthy 8-month-old female child, who presented with features of meningoencephalitis of 2 days' duration. Rapidly moving trophozoites of amoeba were observed in cerebrospinal fluid, which were confirmed to be Naegleria fowleri on polymerase chain reaction. Broad-spectrum antimicrobial therapy with ceftriaxone, vancomycin, amphotericin B and acyclovir was initiated. However, the patient deteriorated and left the hospital against medical advice. The isolation of N. fowleri in this case demands for increased awareness for prompt diagnosis and management in view of its high mortality.

Primary Amebic Meningoencephalitis: A Case Report

Primary amebic encephalitis (PAM) is a devastating central nervous system infection caused by Naegleria fowleri, a free-living amoeba, which can survive in soil and warm fresh water. Here, a 43-year-old healthy male was exposed to warm freshwater 5 days before the symptom onset. He rapidly developed severe cerebral edema before the diagnosis of PAM and was treated with intravenous conventional amphotericin B while died of terminal cerebral hernia finally. Comparing the patients with PAM who has similar clinical symptoms to those with other common types of meningoencephalitis, this infection is probably curable if treated early and aggressively. PAM should be considered in the differential diagnosis of purulent meningoencephalitis, especially in patients with recent freshwater-related activities during the hot season.

Acanthamoeba spp. y Naegleria spp. aisladas del Río Pamplonita, zona metropolitana de Cúcuta, Colombia / Acanthamoeba spp. and Naegleria spp. isolated from Pamplonita river, metropolitan area of Cucuta, Colombia

Introducción: Acanthamoeba y Naegleria son géneros de amebas de vida libre resistentes a cambios extremos de temperatura y pH, aislados de diversos ambientes (suelo, aire y agua). Debido a la cantidad de habitantes que se benefician de las aguas del Río Pamplonita, al desconocimiento de su presencia en la región y al aumento de enfermedades relacionadas con el consumo de agua, se considera necesario realizar estudios sobre la distribución de estos microorganismos. Objetivo: Identificar Acanthamoeba spp. y Naegleria spp. en aguas del curso principal del río Pamplonita de la zona metropolitana de Cúcuta, Colombia. Métodos: La búsqueda de las amebas de interés se realizó mediante examen directo y cultivo a 28, 37 y 42 °C en agar no nutritivo a partir de 21 muestras de agua de siete sectores del río Pamplonita. Adicionalmente, se midió pH y temperatura in situ y se determinó la carga de coliformes. Resultados: Se encontró que 76,2 por ciento de las muestras fueron positivas para alguna de las amebas. La Acanthamoeba fue la que se aisló con mayor frecuencia. El 28,6 por ciento de los cultivos incubados a 42 °C fueron positivos principalmente para Naegleria spp., lo que indica que estos aislados termotolerantes podrían tener potencial patógeno. Conclusiones: Acanthamoeba spp. y Naegleria spp. son frecuentes en aguas del Río Pamplonita en áreas de importante intervención humana. El hallazgo de amebas termotolerantes alerta sobre el riesgo de salud para la población que se encuentra expuesta a esta fuente hídrica(AU)

Introduction: Acanthamoeba and Naegleria are genus of free-living amoeba (AVL) resistant to extreme changes in temperature and pH, isolated from different environments (soil, air and water). Due to the number of inhabitants that benefit from the waters of Pamplonita River, the lack of knowledge about its presence in the region and the increase of diseases related to water consumption, it is necessary to carry out studies on its distribution. Objective: To identify Acanthamoeba spp and Naegleria spp in the water of the main course of Pamplonita River, in the metropolitan area of Cúcuta, Colombia. Methods: The search for the amoebas of interest was performed by direct examination and culture at 28, 37 and 42 °C on non-nutritive agar from 21 water samples from seven sectors of Pamplonita River. Additionally, pH and temperature were measured in situ and the coliforms´ load was also determined. Results: It was found that 76.2 percent of the samples were positive for some of the amoebas, with Acanthamoeba being the most frequently isolated. 28.6 percent of the cultures incubated at 42 °C were positive mainly for Naegleria spp, indicating that these thermotolerant isolates could have pathogen potential. Conclusions: Acanthamoeba spp and Naegleria spp are frequent in waters of Pamplonita River which are areas of important human intervention. The finding of thermotolerant amoebas warns about the health risk for the population that is exposed to this water source(AU)

Monitoring of Noxious Protozoa for Management of Natural Water Resources

Waterborne parasitic protozoa, particularly Giardia lamblia and Cryptosporidium spp., are common causes of diarrhea and gastroenteritis worldwide. The most frequently identified source of infestation is water, and exposure involves either drinking water or recreation in swimming pools or natural bodies of water. In practice, studies on Cryptosporidium oocysts and Giardia cysts in surface water are challenging owing to the low concentrations of these microorganisms because of dilution. In this study, a 3-year monitoring of Cryptosporidium parvum, Giardia lamblia, and Naegleria fowleri was conducted from August 2014 to June 2016 at 5 surface water sites including 2 lakes, 1 river, and 2 water intake plants. A total of 50 water samples of 40 L were examined. Cryptosporidium oocysts were detected in 22% of samples and Giardia cysts in 32%. Water at the 5 sampling sites was all contaminated with Cryptosporidium oocysts (0–36/L), Giardia cysts (0–39/L), or both. The geometric mean concentrations of Cryptosporidium and Giardia were 1.14 oocysts/L and 4.62 cysts/L, respectively. Thus, effective monitoring plans must take into account the spatial and temporal parameters of contamination because they affect the prevalence and distribution of these protozoan cysts in local water resources.

Efficient Liquid Media for Encystation of Pathogenic Free-Living Amoebae

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page’s amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

Amebas de Vida Libre aisladas en aguas superficiales del municipio de Turbaco, Bolívar-Colombia / Free-living amoebae isolated from surface water in the municipality of Turbaco, Bolívar, Colombia

Introducción: las amebas de vida libre (AVL) han sido aisladas de hábitat incluyendo: agua, suelo y vegetación. Los cambios ecológicos y la incursión de estos hábitats, han hecho que puedan invadir un hospedero y vivir como parásitos dentro de él. Por esto, las especies de los géneros Acanthamoeba y Naegleria han sido asociadas con infecciones oportunistas. Objetivo: evaluar la presencia de las amebas de vida libre en fuentes de agua natural en el municipio de Turbaco, con el fin de que sean consideradas en la conducta diagnóstica y terapéutica por las entidades de salud pertinentes. Métodos: se realizó un estudio descriptivo, transversal en los arroyos Matute, Mameyal y Cucumán del municipio de Turbaco - Bolívar. La identificación se hizo mediante el estudio de los frescos de las fuentes de agua observando características morfológicas de las amebas. Resultados: de un total de 54 muestras se obtuvo una positividad del 55,5 por ciento para una o más AVL. Con mayor frecuencia Naegleria sp con un 44,4 por ciento y Acanthamoeba sp, en un 7,4 por ciento. Además, se encontraron otros microorganismos responsables de parasitosis intestinales como: Giardia intestinalis, Blastocystis hominis y Retortomonas intestinalis. Conclusión: los arroyos estudiados son hábitat de las AVL, demostrando que están presentes en el ambiente y que son frecuentes en zonas donde el ser humano suele practicar ciertas actividades, sin embargo, la proporción de Naegleria sp. permite alertar aún más a la población puesto que esta ameba puede afectar a todo tipo de individuo independiente de su estado inmunológico(AU)

Introduction: free-living amoebae (FLAs) have been isolated from habitats such as water, soil and vegetation. As a result of ecological changes and incursion into these habitats, free-living amoebae have invaded hosts and settled within them as parasites. This is the reason why the species of genera Acanthamoeba and Naegleria have been associated with opportunistic diseases. Objective: determine the presence of free-living amoebae in natural water sources from the municipality of Turbaco, so they are taken into account by the corresponding health institutions in the design of diagnostic and therapeutic actions. Methods: a descriptive cross-sectional study was conducted of streams Matute, Mameyal and Cucumán in the municipality of Turbaco, Bolívar. Identification was based on the study of fresh water from the sources selected and observation of the morphological characteristics of amoebae. Results: of the total 54 samples, 55.5 percent tested positive for one or more FLAs. The most common were Naegleria sp with 44.4 percent and Acanthamoeba sp with 7.4 percent. Microorganisms were also found which cause intestinal parasitic disease, such as Giardia intestinalis, Blastocystis hominis and Retortomonas intestinalis. Conclusion: the streams studied are habitats for FLAs, showing that the latter are present in the environment and are common in areas where human beings perform certain activities. However, the proportion of Naegleria sp. makes it necessary to emphatically alert the population, since this amoeba may affect all individuals, irrespective of their immune status(AU)

Prompt diagnosis and extraordinary survival from Naegleria fowleri meningitis: A rare case report.

Primary amoebic meningoencephalitis is a rare fatal meningitis caused by free living amoeba Naegleria fowleri, found in freshwater ponds and lakes. It infects children and young adults with exposure due to swimming or diving. We report a case of N. fowleri meningitis in a 6‑year‑old boy who presented with signs and symptoms of acute bacterial meningitis. No history of travelling or swimming was present. However, the boy frequently played with water stored from a “kuhl” (diversion channels of water). Wet mount of cerebrospinal fluid (CSF) revealed amoeboid and actively motile flagellate forms of trophozoites. CSF culture done on 1.5% non‑nutrient agar plates with a lawn culture of Escherichia coli kept at 37°C for 15 days did not reveal any growth. The test of flagellation on passing CSF in distilled water was however positive in 3 h. Water of the “kuhl” from the stored tank also showed actively motile trophozoites similar to the forms obtained from the CSF. Based on our reports, the boy was immediately treated with amphotericin B, rifampicin and fluconazole for 21 days. Repeat CSF examination after 14 days did not reveal any trophozoites in wet mount and patient was discharged after 3 weeks of successful treatment.

A Fatal Case of Naegleria fowleri Meningoencephalitis in Taiwan

After bathing at a hot spring resort, a 75-year-old man presented to the emergency department because of seizure-like attack with loss of conscious. This is the first case of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri in Taiwan. PAM was diagnosed based on detection of actively motile trophozoites in cerebrospinal fluid using a wet-mount smear and the Liu's stain. The amoebae were further confirmed by PCR and gene sequencing. In spite of administering amphotericin B treatment, the patient died 25 days later.

Demostración del ameboflagelado Naegleria fowleri como agente etiológico de meningoencefalitis amébica primaria en Santiago de Cuba / Demonstration of flagellum ameba Naegleria fowleri as etiological agent of primary amebic meningoencephalitis in Santiago de Cuba

En los últimos años se ha demostrado que el amebo-flagelado Naegleria fowleri es el causante de la meningoencefalitis amébica primaria (MEAP). En Cuba ha sido mencionada la sospecha de su presencia en un líquido céfalo-raquídeo (LCR) de una ameba cuya locomoción y circunstancias sugería que pudiera ser esta ameba. Se realizó el análisis en fresco y en cultivo, con Test de Flagelación de 64 muestras de LCR seleccionadas al azar a partir de las 173 que presentaron las características de transparencia y negatividad a bacterias, requisitos iniciales en búsqueda de este agente, partiendo de una muestra inicial de 1 488 muestras de LCR procedentes de pacientes con meningoencefalitis clínicamente establecidas. De estos 64 LCR, 2 resultaron positivos a la evaluación por los diferentes métodos lo que permitió teñirlos con Giemsa, demostrar su capacidad de flagelación y su resistencia a TMT. Las características citoquímicas de los líquidos concordaban con lo planteado en la literatura y uno de los pacientes sobrevivió, el otro falleció. La patogenicidad de los ameboflagelados aislados de esas dos muestras de LCR se comprobó en el modelo animal ratón albino suizo y fueron observados al microscopio electrónico. Lo anterior confirma microbiológicamente por primera vez en el país este agente etiológico, aunque ya había elementos clínicos y de laboratorio de sospecha en casos anteriores

During the last years it has been demonstrated that the flagellae-carring ameba Naegleria fowleri is the aethiologic agent of the primary amoebic meningoencephalitis (PAME), in Cuba the suspicion of its presence has been mentioned in a Cefalo Raquideal Fluid (CRF) of an ameba whose locomotion and circumstances suggested that it could be this organism. The present work carries out the analysis in fresh CRF samples and after culture, performing the Flagelation Test of the 64 CRF that presented the characteristics of transparency and negativity to bacterial culture ,needed to suspicion of this flagellae-carring ameba, leaving of an starting sample of 1488 CRF.From this analysed sample of CRF only two were positive to the observation of amebas what allowed to tint them with Giemsa and also with Lugol, to demonstrate their flagelation capacity and their resistance to TMT in the corresponding test. The citochemical characteristics of the liquids agreed with that outlined in the literature for this organisms,and one of the patients survived, the other one died. The patogenicity of the isolated flagellae-carring amebas was proven in the animal model of Swiss albino mouse and both isolated strains were observed by the electronic microscope. This results are the basis for considering the present report the first one confirming N.Fowleri as aetiologic agent in two cases of PAME in Cuba

Cepas de Naegleria aisladas en santiago de Cuba y estudiadas a través del microscopio electrónico de barrido / Naegleria strains isolated in Santiago de Cuba and studied using the scanning electron microscope

A partir de 4 localidades donde se aislaron amebas del género Naegleria fowleri en Santiago de Cuba, se realizaron las técnicas necesarias para su adecuado estudio a través del microscopio electrónico de barrido. Con anterioridad, esas amebas fueron inoculadas en biomodelos experimentales (ratones), lo cual permitió determinar un nivel de patogenicidad diferente; y esto último, como es un aspecto controvertido en diversas teorías sobre sus causas (algunas analizadas aquí), entre las que se incluye la sustentada en la invaginación de su superficie (amebostomas o suctores), la autora de este trabajo se dio a la tarea de valorar la presencia (número y características) de los amebostomas o su ausencia a través del mencionado aparato, de donde se derivó que los ameboflagelados no patógenos apenas se diferenciaban de los que sí lo eran. Tal hallazgo pudo ser reafirmado cuando estos se compararon con 2 cepas reconocidas en el Instituto de Medicina Tropical de Checoslovaquia; por consiguiente, ese resultado descarta la citada causa en la patogenicidad de las amebas.

Taking into account 4 localities where amebas of the genus Naegleria fowleri were isolated in Santiago de Cuba, necessary techniques for their adequate study using the scanning electron microscope were performed. Previously, those amebas were inoculated in experimental biomodels (mice), which allowed to determine a different level of pathogenicity, a controversial issue in several theories about their causes (some discussed here), including that maintained in their surface invagination (amebostomas or suckers). Thus, the author of this work evaluated the presence (number and characteristics) or absence of amebostomas through such device, and it is concluded that pathogenic ameboflagellates were similar to those that were not. This finding could be confirmed when they were compared with 2 strains recognized in the Institute of Tropical Medicine of Czechoslovakia. Consequently, that result rules out the above cause in the pathogenesis of amebas.

Amibas de vida libre Acanthamoeba, Balamuthia, Naegleria, Sappinia y Paravahlkampfia: agentes de infección en humano y transmisoras de microorganismos / Free-living amoebae Acanthamoeba, Balamuthia, Naegleria, and Paravahlkampfia Sappinia: agents of human infection and transmission of microorganisms

Se han reportado como agentes causales de enfermedad en humanos: Naegleria fowleri, agente causal de meningoencefalitis amibiana primaria en individuos inmunocompetentes: varias especies de género Acanthamoeba que pueden producir encefalitis granulomatosa amibiana en individuos inmunosuprimidos, úlceras corneales, lesiones en piel y otros órganos en personas inmunocompetentes; Ballamuthia mandrillares agente causal de encefalitis y lesiones en piel en personas inmunocompetentes, Sappinia pedata responsable de un caso de encefalitis en un individuo inmunocompetente y Paravahlkamphfia sp en un caso de queratitis y Paravahlkamphfia francinae en un caso de meningoencefalitis aminiana primera, en paciente en paciente inmunocompetente. No se conoce totalmente los factores de los que depende que las enfermedades producidas por amibas de vida libre se presenten, influyen la presencia de algunas enfermedades, contacto con aguas en las cuales se encuentran estas amibas, y la presencia de enzimas capaces de ejercer efecto citopático. Además el uso de lentes de contacto, algunas costumbres y necesidades humanos rompen el equilibrio ecológico, aumentando así la probabilidad de contacto del hombre con estas amibas. Se han evidenciado en ellas microorganismo en un proceso de endosimbiosis lo que por lo que amibas de vida libre intervienen en la transmisión de ciertos microorganismos patógenos y las amibas se transforman en un incubador potencial y transmisorasde agentes infecciosos. Debe investigarse sobre aspectos biológicos y la relación con el humano.

Have been reported as causative agents of disease in humans: Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis in immunocompetent individuals, several species of Acanthamoeba that con cause granulomatous amoebic encephalitis in immunosuppressed individuals, corneal ulcers, skin lesions and other organs in people immunocompetent; Ballamuthia madrillares causative agent of encephalitis and skin lesions in immunocompetent persons, Sappinia pedata, responsible for a case of encephalitis in an immunocompetent individual Paravahlkamphfia sp in a case of keratitis and Paravanhlkamphfia francinae in a case of primary amoebic meningoencephalitis in an immunocompetent patient. Not fully understood the factors that it depends on diseases caused by free-living amoebae are present, influencing the presence of some diseases, contact with water where are these amoebae, and the presence of enzymes capable of the cytopathic effect. Furthermore, the use of contact lenses, some customs and human needs break the ecological balance, thereby increasing the likelihood of human contact with these amoebae. Have been evidenced in these microorganisms in the process of endosymbiosis that so the free-living amoebae in the transmission of certain pathogenic microorganisms and amoebas and become a potential incubator and transmission of infectious agents. Biological aspects should be investigated and the relationship with the human.

Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-alpha, IL-6, and IL-1beta, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

The nfa1 Gene Contributed on the Contact-dependent Pathogenic Mechanisms of Naegleria fowleri / 한양의대학술지

Free-living Naegleria fowleri is a causal agent of primary amoebic meningoencephalitis in mainly children and young adults. An nfa1 gene, encoding 360 bp of nucleotides, was cloned from a N. fowleri cDNA library by SEREX method. By immunohistochemistry and a confocal microscope, Nfa1 protein was found in amoebic pseudopods, especially in food-cups, when amoeba was in contact with target cells. When an anti-Nfa1 antibody was added to the coculture system, the cytotoxicity of N. fowleri trophozoites onto target cells was decreased, and the severe morphological destruction of rat microglial cells cocultured with N. fowleri trophozoites was reduced. In a tansfection system, an expression vector with an nfa1 gene was successful transfected into nonpathogenic N. gruberi, and transgenic N. gruberi showed the increasing in vitro cytotoxicity. The siRNA decreased the expression levels of nfa1 mRNA and Nfa1 protein in transfected N. fowleri trophozoites. On the immunization of mice with the rNfa1 protein, the protective immunity of host was induced. Thus, mice showed the prolonged mean survival times in PAM-developed mice. In final, the nfa1 gene and Nfa1 protein play an important role in the pathogenesis of N. fowleri infection.