Various brain-eating amoebae: the protozoa, the pathogenesis, and the disease / 医学前沿

Among various genera of free-living amoebae prevalent in nature, some members are identified as causative agents of human encephalitis, in which Naegleria fowleri followed by Acanthamoeba spp. and Balamuthia mandrillaris have been successively discovered. As the three dominant genera responsible for infections, Acanthamoeba and Balamuthia work as opportunistic pathogens of granulomatous amoebic encephalitis in immunocompetent and immunocompromised individuals, whereas Naegleria induces primary amoebic meningoencephalitis mostly in healthy children and young adults as a more violent and deadly disease. Due to the lack of typical symptoms and laboratory findings, all these amoebic encephalitic diseases are difficult to diagnose. Considering that subsequent therapies are also affected, all these brain infections cause significant mortality worldwide, with more than 90% of the cases being fatal. Along with global warming and population explosion, expanding areas of human and amoebae activity in some regions lead to increased contact, resulting in more serious infections and drawing increased public attention. In this review, we summarize the present information of these pathogenic free-living amoebae, including their phylogeny, classification, biology, and ecology. The mechanisms of pathogenesis, immunology, pathophysiology, clinical manifestations, epidemiology, diagnosis, and therapies are also discussed.

Meningoencephalitis due to Naegleria fowleri in cattle in southern Brazil / Meningoencefalite por Naegleria fowleri em um bovino no sul do Brasil

Abstract Naegleria fowleri is a free-living amoeba commonly found in the environment, mainly in fresh water and soil. This protozoon is occasionally involved in cases of fatal central nervous system disease in humans and other animal species. We describe here a case of meningoencephalitis due to Naegleria fowleri in cattle, in southern Brazil. A four-year-old Angus cow presented a clinical history of initial mild neurological signs that progressed to paddling movements, opisthotonus and lateral recumbency after five days. This animal had been kept in an irrigated rice stubble paddock. Grossly, the main lesions consisted of multiple areas of malacia in the right olfactory bulb, piriform lobes, hippocampus, frontal lobe cortex and fornix, along with severe thickening of the mesencephalon and rhombencephalon leptomeninges. Microscopically, severe multifocal necrosuppurative and hemorrhagic meningoencephalitis associated with a large quantity of amoebic trophozoites was present. The latter were confirmed to be Naegleria spp., through immunohistochemistry. Based on the strong congruence with the histopathological data of known cases reported in the literature, a probable association with Naegleria fowleri was established. To our knowledge, this is only the second report of Naegleria fowleri-associated meningoencephalitis in cattle in South America, and it is the first in southern Brazil.

Resumo Naegleria fowleri é uma ameba de vida livre, comumente encontrada no meio ambiente, principalmente em água doce e no solo. Este protozoário é ocasionalmente associado a casos fatais de doença do sistema nervoso central em seres humanos e espécies animais. No presente trabalho, um caso de meningoencefalite por Naegleria fowleri em um bovino na região sul do Brasil é descrito. Uma vaca Angus, de quatro anos de idade apresentou histórico clínico caracterizado inicialmente por sinais neurológicos leves que progrediram para movimentos de pedalagem, opistótono e decúbito lateral após cinco dias. Este animal era mantido em um piquete em resteva de arroz irrigado. Macroscopicamente, as principais lesões foram caracterizadas por múltiplas áreas de malacia no bulbo olfatório direito, lobos piriformes, hipocampo, córtex do lobo frontal e no fórnix, bem como acentuado espessamento das leptomeninges do mesencéfalo e rombencéfalo. Microscopicamente, meningoencefalite necrossupurativa e hemorrágica associada à grande número de trofozoítos amebianos foram observadas. Estes foram confirmados como Naegleria spp. através de imuno-histoquímica. Baseado na forte congruência apresentada entre os dados histopatológicos provenientes de casos conhecidos publicados na literatura, uma provável associação com Naegleria fowleri foi estabelecida. O presente trabalho trata-se do segundo relato de meningoencefalite associada à Naegleria fowleri em bovinos na América do Sul e o primeiro na região sul do Brasil.

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.

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.

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.

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.

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.

Primary amoebic meningoencephalitis: first reported case from Rohtak, North India

A fatal case of primary amoebic encephalitis (PAM) in a 20 year old boy, a proven case of acute leukemic leukemia (ALL) type L2, in remission is described. No history of swimming could be elicited. The clinical presentation, the isolation of the amoeba from the cerebrospinal fluid (CSF), the poor response to amphotericin B, and the ultimately fatal outcome are all consistent with the diagnosis of PAM. On the basis of its ability to grow at temperature 42ºC and 45ºC, morphology of trophozoite, and the presence of flagellate forms in CSF, the amoeba was identified as Naegleria fowleri. Other drugs used in combination with amphotericin B are tetracycline, rifampicin, and miconazole. A possibility of PAM should always be considered in all cases of acute purulent meningoencephalitis in which no bacteria or fungus are found.

Primary amebic meningoencephalitis.

We report an 8 month-old infant with primary amebic meningoencephalitis (PAME) due to Naegleria fowleri. The child was treated with amphotericin B, chloramphenicol and rifampicin for 3 weeks. PAME is an almost universally fatal condition with very few survivors till date. Our patient was one of the rare survivors who recovered after treatment and was discharged without any residual neurological deficit.

Primary amoebic meningoencephalitis due to Naegleria fowleri.

Primary amoebic meningoencephalitis (PAM) due to Naegleria fowleri was detected in a 36-year-old, Indian countryman who had a history of taking bath in the village pond. He was admitted in a semi comatosed condition with severe frontal headache, neck stiffness, intermittent fever, nausea, vomiting, left hemiparesis and seizures. Computerized tomography (CT) scan of brain showed a soft tissue non-enhancing mass with erosion of sphenoid sinus. However CSF findings showed no fungal or bacterial pathogen. Trophozoites of Naegleria fowleri were detected in the direct microscopic examination of CSF and these were grown in culture on non-nutrient agar. The patient was put on amphotericin-B, rifampicin and ceftazidime but his condition deteriorated and was taken home by his relatives in a moribund condition against medical advice and subsequently died. A literature review of 7 previous reports of PAM in India is also presented. Four of theses eight cases were non lethal. The mean age was 13.06 years with male: female ratio of 7:1. History of contact with water was present in four cases. Trophozoites could be identified in all 8 cases in this series.

Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.

Meningoencefalitis amebiana primaria: comunicacion de dos nuevos casos Venezolanos / Primary amebic meningoencephalitis: two new cases report from Venezuela

La meningoencefalitis amebiana primaria (MAP) es infrecuente. Describimos dos nuevos casos de MAP en pacientes Venezolanos. Caso 1, Varón de 10 años, con fiebre, cefalea, vómitos y debilidad generalizada, y antecedente de inmersión en un estanque de agua días antes del inicio de sus síntomas, falleciendo 72 horas después del ingreso. Caso 2, Varón de 23 años con historia de cefalea, fiebre, vómitos, somnolencia y cambios de conducta. El paciente falleció 40 horas después. El estudio neuropatológico en ambos casos reveló MAP por Naegleria fowleri. La encefalitis por amebas anfizoicas debe sospecharse en casos de meningoencefalitis asépticas.

Primary amebic menigoencephalitis (PAM) is rare. Two cases of PAM in Venezuelan patients are described. Case 1, a 10 year-old male with headache, fever, vomiting. The patient swam in a water reservoir before the onset of his disease. He died during his third hospital day. Case 2, a 23 year-old male with a history of headache, fever, vomiting, drowsiness, and behavioral disturbances. The patient died on his second hospital day. The diagnosis in both cases was PAM due to Naegleria fowleri. Central nervous system infection by free-living amebas should be considered in meningoencephalitides with bacterial-free cerebro-spinal fluid.

Meningoencephalitis by Naegleria fowleri: epidemiological study in Anzoategui state, Venezuela

Um caso de meningoencefalite amebiana primária, causada por Naegleria fowleri, foi diagnosticada no município de Independência no Estado de Anzoategui, Venezuela. Este caso motivou a realização deste estudo epidemiológico com o objetivo de identificar amebas de vida livre nessa área. Foram colhidas amostras representativas de água e realizadas análises fisicoquímicas e microbiológicas. Trofozoítos e cistos de Naegleria spp foram detectados em 44,4% (n=4). Verificou-se excelente concordância entre a observação das amebas de vida livre em esfregaços e aquelas de culturas monoxênicas em ágar não nutriente com Klebsiella pneumoniae (Kappa=1; p=0,003). Obteve-se uma carga variável de microrganismos mesófilos aeróbicos. As médias de mofos e leveduras foram de 3,0 CFU/ml (SD+2,0) e 102,9 CFU/ml(SD+32,2), respectivamente. Cem por cento das amostras apresentaram um número maior provável de coliformes totais e fecais de 240.000 NMP/100ml. Naegleria spp estava presente nas águas do município de Independência no Estado de Anzoategui, o que constitui um risco para a população que usa essas fontes.

Primary amebic meningoencephalitis

This is the first case description of primary amebic meningoencephalitis [PAM] in an Iranian child. The patient was an eighteen months old boy referred to the hospital with signs and symptoms of acute meningitis. Cerebrospinal fluid [CSF] examination indicated pleocytosis with predominance of neutrophils, low sugar and high protein. Gram staining and culture of CSF for bacteria were negative. Microscopic examination of CSF revealed the motile amebae with pseudopodia. In Giemsa staining of CSF, the trophozoites of amebae were observed. The initial response to a six-week treatment course with amphotericin B, rifampin and chloramphenicol was satisfactory, but in the follow-ups the patient regressed neurologically. Although PAM is a rare and fatal disease, it should be differentiated from bacterial meningitis and treatment must be promptly carried out

Amphizoic amoebae: pathogenic free-living protozoa; review of the literature and review of cases in Thailand.

A large variety of species of free-living amoebae (FLA) caused an indefinite form of these protozoa. Non-fixed form, as indicated by amoeboid movement and possed the bacteria to survive in nature. Two species of pathogenic FLA: Naegleria fowleri and Acanthamoeba spp. were identified as the causative agents of Primary Amoebic Meningoencephalitis (PAM) and Granulomatous Amoebic Encephalitis (GAE) respectively. They were suggested to amphizoic protozoa, capable of living as parasites or as free-living and they were also considered to be distributed worldwide. These amoebae were detected in lakes, rivers and ponds. The first case of meningoencephalitis was observed in 1961 by Fowler Many cases were reported later on and the pathogenicity was tested by nasal inoculation of mice. In fact, quite a number of FLA were isolated but only a few species were pathogenic to humans. The three typical features which allow recognition of Naegleria spp. flagellate stage, round cyst and promitotic trophozoite. This promitosis distinguishes the Naegleria genus from Acanthamoeba spp. The disease caused by PAM usually occurs with acute onset, whereas chronic for GAE. The GAE cases mentioned are mostly in debilitated patients, chronic alcoholics or patients under treatment with immunosuppressive methods. About 6 cases of PAM were reported in Thailand during 1982-1997. Four cases of GAE were reported in 1994 and two isolated cases ofAcanthamoebafrom keratitis patients were reported in 2000. Finally one case of PAM and one case of GAE were reported in 2001. The surveys of FLA were set up to study the distribution of these pathogenic amoebae and determine the prevalence of amoebae in aquatic habitats of human environments. About 40% were identified as Acanthamoeba spp., 30% were Naegleria spp., 20% were Hartmanella and 10% were Vahlkampfia. Only 10% of Naegleria spp. belonged to Naegleria fowleri.