A comparative study of the membrane proteins from Naegleria species: A 23-kDa protein participates in the virulence of Naegleria fowleri.

The plasma membrane is essential in the pathogenicity of several microorganisms. However, to date, there are few studies related to the plasma membrane proteins in Naegleria fowleri; this amoeba produces a fatal disease called primary amoebic meningoencephalitis. In the present study, we analyzed the electrophoretic pattern of the membrane proteins of N. fowleri and compared it with the nonpathogenic N. lovaniensis and N. gruberi. We detected a 23-kDa protein (Nf23) present at a higher level in N. fowleri than in the nonpathogenic amoebae. The mass spectrometry analysis showed that the Nf23 protein has a sequence of 229 amino acids that corresponds to a membrane protein. The mRNA level of nf23 was overexpressed 4-fold and 40,000-fold in N. fowleri compared with N. lovaniensis and N. gruberi, respectively. Moreover, we found a 5-fold overexpression of nf23 in N. fowleri trophozoites recovered from mouse brains compared with trophozoites axenically cultivated. In addition, the cytopathic effect on Madin-Darby Canine Kidney cells coincubated with N. fowleri diminished in the presence of antibodies against Nf23; nevertheless, the nonpathogenic amoebae did not produce damage to the monolayer cells. These results suggest that the plasma membrane protein Nf23 is probably involved in the virulence of N. fowleri.

What do we know by now about the genus Naegleria?

In this short overview of the genus Naegleria a brief historical sketch is given since the discovery of this amoeboflagellate in 1899 and the finding in 1970 that one species, Naegleria fowleri causes primary amoebic meningoencephalitis in man. Eight different types of this pathogen are known which have an uneven distribution over the world. Until now 47 different Naegleria spp. are described, of which two other species cause disease in experimental animals, and their geographical dispersal is indicated. The presence of group I introns in the SSU and in the LSU rDNA in the genus is discussed, as well as the possibility of sex or mating. It is also mentioned that the genome of N. fowleri should not be compared to that of Naegleria gruberi, to know why the former is pathogenic, but to the genome of its closest relative Naegleria lovaniensis.

Screening of recreational areas of rivers for potentially pathogenic free-living amoebae in the suburbs of Tehran, Iran.

A survey was conducted to determine the presence of free-living amoebae (FLA), especially Acanthamoeba and Naegleria, in river recreation areas in Tehran Province, Iran. All rivers surveyed were associated with human activity, and two were also a source of municipal tap water. Fifty-five water samples from 10 major rivers were screened for FLA and identified by morphological characters, PCR amplification targeting specific genes for Acanthamoeba (DF3 region of Rns gene) and other FLA (ITS PCR), and homology analysis. The percentage of positive FLA isolates was 27.3%, of which 80% were Acanthamoeba, assigned to the T4 and T15 genotype, and 20% were Naegleria. Isolation of Acanthamoeba T4 genotype (91.7%) from recreation areas could be a health threat and a sanitary risk associated with human activity where young people and tourists congregate in summer. Posting of warning signs and education of high-risk individuals are important for disease prevention. To the best of our knowledge this is the first report of genotype T15 (clustered with A. jacobsi) identified in Iran and the first report of the distribution of FLA such as Naegleria (N. pagei, N. clarki and N. fultoni) in recreation areas in rivers of Tehran Province using molecular methods.

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

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(AU)

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(AU)

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.

Differences between Naegleria fowleri and Naegleria gruberi in expression of mannose and fucose glycoconjugates.

Naegleria fowleri is the etiologic agent of primary amoebic meningoencephalitis, a rapidly fatal parasitic disease of humans. The adherence of Naegleria trophozoites to the host cell is one of the most important steps in the establishment and invasiveness of this infectious disease. Currently, little is known about the surface molecules that may participate in the interaction of N. fowleri with their target cells. In the present study, we investigated the composition of glycoconjugates present on the surface of trophozoites of the pathogenic N. fowleri and the nonpathogenic Naegleria gruberi. With the use of biotinylated lectins in western blot and flow cytometric analysis, we showed that N. fowleri trophozoites present high levels of surface glycoconjugates that contain alpha-D-mannose, alpha-D-glucose, and terminal alpha-L-fucose residues. A significant difference in the expression of these glycoconjugates was observed between N. fowleri and the nonpathogenic N. gruberi. Furthermore, we suggest that glycoconjugates that contain D-mannose and L-fucose residues participate in the adhesion of N. fowleri and subsequent damage to MDCK cells.

Prevalence of potentially pathogenic free-living amoebae from Acanthamoeba and Naegleria genera in non-hospital, public, internal environments from the city of Santos, Brazil

Acanthamoeba and Naegleria species are free-living amoebae (FLA) found in a large variety of natural habitats. The prevalence of such amoebae was determined from dust samples taken from public non-hospital internal environments with good standards of cleanliness from two campuses of the same University in the city of Santos (SP), Brazil, and where young and apparently healthy people circulate. The frequency of free-living amoebae in both campuseswas 39 percent and 17 percent respectively, with predominance of the genus Acanthamoeba. On the campus with a much larger number of circulating individuals, the observed frequency of free-living amoebae was 2.29 times larger (P< 0.00005). Two trophozoite forms of Naegleria fowleri, are the only species of this genus known to cause primary amoebian meningoencephalitis, a rare and non-opportunistic infection. We assume that the high frequency of these organisms in different internal locations represents some kind of public health risk.

Prevalence of potentially pathogenic free-living amoebae from Acanthamoeba and Naegleria genera in non-hospital, public, internal environments from the city of Santos, Brazil.

Acanthamoeba and Naegleria species are free-living amoebae (FLA) found in a large variety of natural habitats. The prevalence of such amoebae was determined from dust samples taken from public non-hospital internal environments with good standards of cleanliness from two campuses of the same University in the city of Santos (SP), Brazil, and where young and apparently healthy people circulate. The frequency of free-living amoebae in both campuses was 39% and 17% respectively, with predominance of the genus Acanthamoeba. On the campus with a much larger number of circulating individuals, the observed frequency of free-living amoebae was 2.29 times larger (P< 0.00005). Two trophozoite forms of Naegleria fowleri, are the only species of this genus known to cause primary amoebian meningoencephalitis, a rare and non-opportunistic infection. We assume that the high frequency of these organisms in different internal locations represents some kind of public health risk.

Rapid, sensitive, and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis.

The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

Early detection and identification of amphizoic amoebae from nasal exudates of a symptomatic case.

A man visited the Out Patient Department of the hospital for Tropical Diseases in February 2004 with low grade fever and severe headache for a week. He had the history of diving in a natural pond 2-3 days before the onset of the disease. A thick bloody mucous was observed from the nasal discharge. Fresh microscopic observation of the exudates in 0.85% sodium chloride revealed numerous active amoeba trophozoites. Two groups of the trophozoites were observed The first group was 10 micro sized amoeba with active directional movement by lobopodia and the second group was 15-30 micro sized amoeba with active multiprogressive movement by filopodia. Few flagellate forms were observed after exflagellation in distilled water and some polygonal cysts were also found. Giemsa' stain was used to differentiate the amoeba trophozoites from the leukocytes. It was concluded that this patient was infected by both Naegleria spp. and Acanthamoeba spp. This is the first report of double infection of free-living amoeba in a symptomatic and non-fatal patient.

Free-living ameba contamination in natural hot springs in Thailand.

Thermo tolerant free-living ameba, Naegleria spp and Acanthamoeba spp contamination in natural hot springs in Thailand were carried out from 13 provinces. The temperature of hot springs water varied from 28 degrees-65 degrees C and pH from 6-8. We found that 38.2 % (26/68) of water samples were positive, Acanthamoeba was 13.2% (9/68) whilst Naegleria was 35.3% (24/68). Contamination by free-living ameba in natural hot springs may pose a significant health risk to people who use such water for recreation activities.

Spa, springs and safety.

Natural mineral water has long been used worldwide for bathing and health purposes. At present, Thailand is famous for health spas and natural hot springs among local people and tourists. Due to possible risks of exposure to harmful agents, we studied hazardous pollutants at 57 natural hot springs from 11 provinces in northern, central, eastern and southern Thailand. Pathogenic, free-living amebae of the genera Naegleria and Acanthamoeba, which can cause central nervous system infection, were found in 26.3% (15/57) and 15.8% (9/ 57), respectively. Dissolved radon, a soil gas with carcinogenic properties, was present in nearly all hot springs sites, with concentration ranging from 0.87-76,527 Becquerels/m3. There were 5 water samples in which radon concentration exceeded the safety limit for drinking. Legionella pneumoniphila (serogroups 1, 3, 5, 6, 7 10 and 13) were found in samples from 71.9% (41/57) of studied sites. Because spas and natural springs are popular tourist attractions, health authorities should be aware of possible hazards and provide tactful measures and guidelines to ensure safety without causing undue alarm to foreign and Thai tourists.

Pathogenic free-living amoebae in Korea.

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.

Detection and significance of the potentially pathogenic amoeboflagellate Naegleria italica in Australia.

Thermophilic amoeboflagellates in the genus Naegleria include both virulent and benign species. One of the less studied species, N. italica, has not been detected in the environment since the first reports from Italy in the 1980s; its virulence is known only from infection of laboratory mice. Two recent strains from recreational water in Western Australia (AWQC NG960, NG961) were tentatively identified as N. italica from the characteristic mobilities of seven isozymes. Sequences of the 5.8S rRNA gene and its flanking ITS aligned with a 380+bp length of the published sequence for N. italica with 98% identity. Differences from the type strain were confined to ITS2. Shorter alignments (<320 bp) were observed with other Naegleria species, corresponding to conserved regions of the 5.8S gene and ITS. Unlike the European type strain of N. italica, the Australian isolates failed to infect laboratory mice intranasally, confirming that infectivity of this species is variable and often lower than in N. fowleri.

Effect of antifungal drugs on pathogenic Naegleria spp isolated from natural water sources.

UNLABELLED: Five of 16 strains of pathogenic Naegleria spp isolated from 350 natural water sources in Taling Chan District, Bangkok had similar molecular weights and zymogram patterns to those of Naegleria fowleri CDCVO 3081 and Thai strain. The in vitro effects of antifungal drugs (amphotericin B, ketoconazole, fluconazole and itraconazole) were tested at the following concentrations: amphotericin B 0.01-0.55 microg/ml, ketoconazole 0.01-0.3 microg/ml, fluconazole 0.75-3.5 mg/ml and itraconazole 4-12 mg/ml respectively. Aliquots (15,000 cells/ml) of the amoebae were placed in the cells of the microtiter plate and incubated at 37 degrees C. Amoebae from each treatment sample were exposed to one of the four antifungal drugs. Statistical analysis was done by dependent t-test. The sensitivity of the antifungal drugs (MIC50) was as follows: amphotericin B 0.03-0.035 microg/ml ketoconazole 0.05-0.15 microg/ml fluconazole 1.75 mg/ml and itraconazole 8-9 mg/ml respectively (p < 0.005). CONCLUSION: Amphotericin B and ketoconazole are more active against Naegleria fowleri in vitro. The results of the present study should be used as an in vitro screening test for drugs that have potential amebicidal activity.

PCR detection and analysis of the free-living amoeba Naegleria in hot springs in Yellowstone and Grand Teton National Parks.

Free-living thermotolerant amoebae pose a significant health risk to people who soak and swim in habitats suitable for their growth, such as hot springs. In this survey of 23 different hot springs in Yellowstone and Grand Teton National Parks, we used PCR with primer sets specific for Naegleria to detect three sequence types that represent species not previously described, as well as a fourth sequence type identified as the pathogen Naegleria fowleri.

December 2002: 19-year old male with febrile illness after jet ski accident.

The December 2002 COM. A 19-year-old healthy male fell into stagnant water of the intercostal waterway (salt water of South Florida), following a jet ski accident. He sustained minor superficial injuries but engulfed significant quantities of water and sediment. A few days later he developed bifrontal headaches, vomiting, a stiff neck and a temperature of 102 degrees F. A CT scan on admission without contrast was negative. The CSF had markedly elevated white count but bacterial and fungal cultures were negative. He became progressively lethargic. On the fifth day he developed seizure activity. He expired the next day despite antibiotics. Gross examination of the brain at autopsy revealed edema, cerebellar tonsillar herniation and purulent meningitis. Microscopic examination revealed a massive leptomeningeal inflammatory infiltrate composed of neutrophils, lymphocytes, and numerous histiocyte-like cells. The inflammatory infiltrate extended into the cerebral parenchyma in numerous areas also involving the cerebellum, brainstem and ventricular system. Given the exposure to stagnant water (later confirmed to be a man-made fresh water lake), and the numerous histiocytic-like cells, suspicion for an amebic etiology of the disease process was raised and the CDC identified the ameba as Naegleria Fowleri. Infection by Naegleria Fowleri, a free-living ameba, occurs after exposure to polluted water in man-made fresh water lakes, ponds, swimming pools, particularly during the warm weather months when the thermophilic ameba grows well. The pathologic substrate of the infection is an acute hemorrhagic, necrotizing meningo-encephalitis mainly at the base of the brain, brainstem and cerebellum occurring in young, healthy individuals.

[Isolation of potencially pathogenic free-living amoebas in hospital dust]. / Isolamento de amebas de vida livre potencialmente patogênicas em poeira de hospitais.

OBJECTIVE: To evaluate the occurrence of free-living amoebas of the genera Acanthamoeba and Naegleria is dust samples colleted in two hospitals. METHODS: One-hundred and thirty-two dust samples were collected in two hospitals in Brazil. Hospital collection sites were the following: intensive care unit, operation rooms, nursery, kitchen, emergency and infectious diseases isolation room. The isolation of the amoebas was performed in three culture media: non-nutrient agar inoculated with Escherichia coli, soy agar, and microculture in Giazzi-modified Pavlova's medium. The amoebas were identified according to morphological criteria. RESULTS: Amoebas of the genera Acanthamoeba and Naegleria were found in 45.5% of the samples, of which 41.6% were collected in the university hospital and 50% in the state hospital. Of all, 45.5% were positive for the genera Acanthamoeba and 3.8% for genera Naegleria. CONCLUSIONS: Potentially pathogenic free-living amoebas were seen in all sites of the two hospitals and Acanthamoeba was the most frequently isolated genera.

Effects of peracetic acid and monochloramine on the inactivation of Naegleria lovaniensis.

Biocidal activities of monochloramine and peracetic acid were studied on cysts of Naegleria lovaniensis. Until recently the most commonly used biocide to disinfect cooling water systems was hypochlorite. Owing to its negative impact on the aquatic environment, ecologically less harmful alternatives have been sought. As the biocidal activity of monochloramine and peracetic acid makes them good candidates for inactivation of pathogenic Naegleria species, these biocides were tested against Naegleria lovaniensis, a relative of the pathogen Naegleria fowleri, as an alternative treatment to hypochlorite. Under laboratory conditions the biocidal activity of hypochlorite was 8- 10x stronger than that of the two investigated substances. Hypochlorite, at a concentration of 0.5 mg/L, killed 100% Naegleria lovaniensis after 1 h exposure (25 degrees C, pH 7.3- 7.4). To achieve similar results with monochloramine and peracetic acid, 3.94 mg/L or 5.33 mg/L had to be used respectively (25 degrees C, pH 8). It was known that the in situ biota of the biofilm, along with any organic material in the water column, had a negative impact on the efficiency of the biocides. There are, however, indications that the relative efficacy of monochloramine and peracetic acid was quite good under such conditions when compared with hypochlorite.