Naegleria

Global incidence of primary amoebic meningoencephalitis cases associated with domestic drinking water is increasing. The need for understanding disinfectant regimes capable of eliminating the causative microorganism, Naegleria fowleri, from bulk water and pipe wall biofilms is critical. This field study demonstrated the successful elimination of N. fowleri from the bulk water and pipe wall biofilm of a persistently colonised operational drinking water distribution system (DWDS), and the prevention of further re-colonisation. A new chlorination unit was installed along the pipe line to boost the free chlorine residual to combat the persistence of N. fowleri. Biofilm and bulk water were monitored prior to and after re-chlorination (RCl), pre-rechlorination (pre-RCl) and post-rechlorination (post-RCl), respectively , for one year. A constant free chlorine concentration of > 1 mg/L resulted in the elimination of N. fowleri from both the bulk water and biofilm at the post-RCl site. Other amoeba species were detected during the first two months of chlorination, but all amoebae were eliminated from both the bulk water and biofilm at post-RCl after 60 days of chlorination with free chlorine concentrations > 1 mg/L. In addition, a dynamic change in the biofilm community composition and a four log reduction in biofilm cell density occurred post-RCl. The pre-RCl site continued to be seasonally colonised by N. fowleri, but the constant free chlorine residual of > 1 mg/L prevented N. fowleri from recolonising the bulk and pipe wall biofilm at the post-RCl site. To our knowledge, this is the first study to demonstrate successful removal of N. fowleri from both the bulk and pipe wall biofilm and prevention of re-colonisation of N. fowleri in an operational DWDS. The findings of this study are of importance to water utilities in addressing the presence of N. fowleri and other amoeba in susceptible DWDSs.

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred worldwide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the hu...

Naegleria fowleri is a small free-living amoebo-flagellate found in natural and manmade thermal aquatic habitats worldwide. The organism is pathogenic to man causing fatal primary amoebic meningoencephalitis (PAM). Infection typically results from bathing in contaminated water and is usually fatal. It is, therefore, important to identify sites containing N. fowleri in the interests of preventive public health microbiology. Culture of environmental material is the conventional method for the isolation of N. fowleri but requires several days incubation and subsequent biochemical or molecular tests to confirm identification. Here, a nested one-step PCR test, in conjunction with a direct DNA extraction from water or sediment material, was developed for the rapid and reliable detection of N. fowleri from the environment. Here, the assay detected N, fowleri in 18/109 river water samples associated with a nuclear power plant in South West France and 0/10 from a similar site in the UK. Although culture of samples yielded numerous thermophilic free-living amoebae, none were N. fowleri or other thermophilic Naegleria spp. The availability of a rapid, reliable and sensitive one-step nested PCR method for the direct detection of N. fowleri from the environment may aid ecological studies and enable intervention to prevent PAM cases. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.

We have recently shown that the ribosomal RNA genes of the amoebo-flagellate Naegleria gruberi Schardinger, 1899, strain NEG-M are carried exclusively on a 14 kilobasepair plasmid. To explore the distribution of this unique gene arrangement, we have examined another strain of N. gruberi and four other species from the order Schizopyrenida. All have this unusual gene arrangement although the size of the plasmid varies widely. Species groups based on morphological criteria do not agree with those resulting from comparison of plasmid restriction enzyme patterns.

Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthanweba and Naegleria for clinical, epidemiological or public health use.

This study reports the detection of Acanthamoeba and Naegleria species in 14 swimming pools around Petaling Jaya and Kuala Lumpur, Malaysia. Sampling was carried out at 4 sites (the platforms (P), wall (W), 1 meter from the wall (1) and middle (2)) of each swimming pool. These free living amoebae (FLA) were detected under light and inverted microscopes after being cultured on the surface of non-nutrient agar lawned with Escherichia coli. Acanthamoeba species were detected in higher number of culture plates from all sampling sites of all the swimming pools. While Naegleria, were detected in fewer culture plates at 3 sampling sites (absent at site P) of 8 swimming pools. This suggested that the thick double-walled cysts of Acanthamoeba were more resistant, thus remaining viable in the dry-hot areas of the platforms and in chlorinated water of the swimming pools whereas Naegleria cysts, that are fragile and susceptible to desiccation, preferred watery or moist areas for growth and proliferation. The prevalence of both FLA was highest at site W (76.2%), followed by site 1 (64.7%), lowest at site 2 (19.4%), and could be detected at all 3 sampling levels (top, middle and bottom) of these 3 sites. The surface of site W might act as a bio-film that accumulated all kinds of microbes providing sufficient requirement for the FLA to develop and undergo many rounds of life cycles as well as moving from top to bottom in order to graze food. Other factors such as human activities, the circulating system which was fixed at all swimming pools, blowing wind which might carry the cysts from surroundings and the swimming flagellate stage of Naegleria, could also contribute to the distribution of the FLA at these sampling sites. Both FLA showed highest growth (80.4%) at room temperature (25-28 degrees C) and lesser (70.0%) at 37 degrees C which might be due to the overgrowth of other microbes (E. coli, fungi, algae, etc). While at 44 degrees C, only Acanthamoeba species could survive thus showing that our swimming pools are free from potentially pathogenic Naegleria species. However, further study is needed in order to confirm the virulence levels of these amoebae isolates.

Lateral gene transfer (LGT) in eukaryotes from non-organellar sources is a controversial subject in need of further study. Here we present gene distribution and phylogenetic analyses of the genes encoding the hybrid-cluster protein, A-type flavoprotein, glucosamine-6-phosphate isomerase, and alcohol dehydrogenase E. These four genes have a limited distribution among sequenced prokaryotic and eukaryotic genomes and were previously implicated in gene transfer events affecting eukaryotes. If our previous contention that these genes were introduced by LGT independently into the diplomonad and Entamoeba lineages were true, we expect that the number of putative transfers and the phylogenetic signal supporting LGT should be stable or increase, rather than decrease, when novel eukaryotic and prokaryotic homologs are added to the analyses. The addition of homologs from phagotrophic protists, including several Entamoeba species, the pelobiont Mastigamoeba balamuthi, and the parabasalid Tricho...

Naegleria is more likely a hazardous condition for the world. It is fulminant and water borne protozoan disease transmitted through warm fresh water environment and infects the human causing Primary Amoebic Meningoencephalitis (PAM). The most common clinical findings regarding naegleria are severe headache, sudden onset of fever, neck stiffness, and altered mental status. However, presently the treatment includes antifungal agent such as Amphotericin B or along with combination of Fluconazole, Miconazole etc. but yet no effective treatment to safe the precious lives. Preventive measures must be effective and reduces the risk factors by using purify water. Although escalate of that disease is rapidly all over the world and also in Pakistan. Our survey aims to find out the awareness about naegleria in the population of Karachi. A cross-sectional and random sampling method was used to conduct data from people belonging to different genders, age groups and occupations. Data from 200 samples were collected and analyzed. According to our survey, 39% people have awareness about naegleria, 71.5% have awareness about transmission, 51.5% have knowledge about the signs and symptoms, and 42.5% thinks that naegleria is curable while 43% people in Karachi have awareness about the prevention and control of naegleria.

Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombinat...

The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis. N. fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined. In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp. before and after thermal additions from a nuclear power plant. Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp. and pathogenic N. fowleri. Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis. N. fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir. The probability of isolating thermophilic Naegleria and pathogenic N. fowleri increased si...

Modified DNA bases in mammalian genomes, such as 5-methylcytosine ((5m)C) and its oxidized forms, are implicated in important epigenetic regulation processes. In human or mouse, successive enzymatic conversion of (5m)C to its oxidized forms is carried out by the ten-eleven translocation (TET) proteins. Previously we reported the structure of a TET-like (5m)C oxygenase (NgTET1) from Naegleria gruberi, a single-celled protist evolutionarily distant from vertebrates. Here we show that NgTET1 is a 5-methylpyrimidine oxygenase, with activity on both (5m)C (major activity) and thymidine (T) (minor activity) in all DNA forms tested, and provide unprecedented evidence for the formation of 5-formyluridine ((5f)U) and 5-carboxyuridine ((5ca)U) in vitro. Mutagenesis studies reveal a delicate balance between choice of (5m)C or T as the preferred substrate. Furthermore, our results suggest substrate preference by NgTET1 to (5m)CpG and TpG dinucleotide sites in DNA. Intriguingly, NgTET1 displays ...

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.

This study reports the detection of Acanthamoeba and Naegleria species in 14 swimming pools around Petaling Jaya and Kuala Lumpur, Malaysia. Sampling was carried out at 4 sites (the platforms (P), wall (W), 1 meter from the wall (1) and middle (2)) of each swimming pool. These free living amoebae (FLA) were detected under light and inverted microscopes after being cultured on the surface of non-nutrient agar lawned with Escherichia coli. Acanthamoeba species were detected in higher number of culture plates from all sampling sites of all the swimming pools. While Naegleria, were detected in fewer culture plates at 3 sampling sites (absent at site P) of 8 swimming pools. This suggested that the thick double-walled cysts of Acanthamoeba were more resistant, thus remaining viable in the dry-hot areas of the platforms and in chlorinated water of the swimming pools whereas Naegleria cysts, that are fragile and susceptible to desiccation, preferred watery or moist areas for growth and proliferation. The prevalence of both FLA was highest at site W (76.2%), followed by site 1 (64.7%), lowest at site 2 (19.4%), and could be detected at all 3 sampling levels (top, middle and bottom) of these 3 sites. The surface of site W might act as a bio-film that accumulated all kinds of microbes providing sufficient requirement for the FLA to develop and undergo many rounds of life cycles as well as moving from top to bottom in order to graze food. Other factors such as human activities, the circulating system which was fixed at all swimming pools, blowing wind which might carry the cysts from surroundings and the swimming flagellate stage of Naegleria, could also contribute to the distribution of the FLA at these sampling sites. Both FLA showed highest growth (80.4%) at room temperature (25-28 degrees C) and lesser (70.0%) at 37 degrees C which might be due to the overgrowth of other microbes (E. coli, fungi, algae, etc). While at 44 degrees C, only Acanthamoeba species could survive thus showing that our swimming pools are free from potentially pathogenic Naegleria species. However, further study is needed in order to confirm the virulence levels of these amoebae isolates.

Ribosomal DNA of several species of the free-living Naegleria amoeba harbors an optional group I intron within the nuclear small subunit ribosomal RNA gene. The intron (Nae.S516) has a complex organization of two ribozyme domains (NaGIR1 and NaGIR2) and a homing endonuclease gene (NaHEG). NaGIR2 is responsible for intron excision, exon ligation, and full-length intron RNA circularization, reactions typical for nuclear group I intron ribozymes. NaGIR1, however, is essential for NaHEG expression by generating the 5' end of the homing endonuclease messenger RNA. Interestingly, this unusual class of ribozyme adds a lariat-cap at the mRNA. To elucidate the evolutionary history of the Nae.S516 twin-ribozyme introns we have analyzed 13 natural variants present in distinct Naegleria isolates. Structural variabilities were noted within both the ribozyme domains and provide strong comparative support to the intron secondary structure. One of the introns, present in N. martinezi NG872, con...

Monoclonal antibodies (MAbs) reactive to the pathogenic amoeba Naegleria fowleri were analyzed by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay, Western blotting (immunoblotting), and radioimmunoprecipitation assay (RIPA). Two MAbs (3A4 and 5D12) showed reactivity by ELISA with all N. fowleri strains tested and no reactivity with the five other Naegleria species, N. lovaniensis, N. gruberi, N. australiensis, N. jadini, and N. andersoni. These MAbs reacted with the three morphological forms of N. fowleri (trophozoites, cysts, and flagellates). The reactivity on Western blots was suppressed by treatment with metaperiodate, suggesting a carbohydrate epitope. Differences in reactivity patterns between trophozoites and cysts observed with radioimmunoprecipitation assay might reflect differences in biological properties. The formalin stability of the epitope may be useful in detecting N. fowleri in fixed biopsies and in investigating the pathological process.

Background: In Malaysia, researchers and medical practitioners are unfamiliar with Naegleria infections. Thus little is known about the existence of pathogenic Naegleria fowleri, and the resultant primary amoebic meningoencephalitis (PAM) is seldom included in the differential diagnosis of central nervous system infections. This study was conducted to detect the presence of Naegleria species in various environmental samples. Methods/Findings: A total of 41 Naegleria-like isolates were isolated from water and dust samples. All these isolates were subjected to PCR using two primer sets designed from the ITS1-ITS2 regions. The N. fowleri species-specific primer set failed to produce the expected amplicon. The Naegleria genus-specific primers produced amplicons of 408 bp (35), 450 bp (2), 457 bp (2) or 381 bp (2) from all 41 isolates isolated from aquatic (33) and dust (8) samples. Analysis of the sequences from 10 representative isolates revealed that amplicons with fragments 408, 450 and 457 bp showed homology with nonpathogenic Naegleria species, and 381 bp showed homology with Vahlkampfia species. These results concurred with the morphological observation that all 39 isolates which exhibited flagella were Naegleria, while 2 isolates (AC7, JN034055 and AC8, JN034056) that did not exhibit flagella were Vahlkampfia species. Conclusion: To date, pathogenic species of N. fowleri have not been isolated from Malaysia. All 39 isolates that produced amplicons (408, 450 and 457 bp) from the genus-specific primers were identified as being similar to nonpathogenic Naegleria. Amplicon 408 bp from 5 representative isolates showed 100% and 99.7% identity to Naegleria philippinensis isolate RJTM (AM167890) and is thus believed to be the most common species in our environment. Amplicons 450 bp and 457 bp were respectively believed to be from 2 new species of Naegleria, since representative isolates showed lower homology and had a longer base pair length when compared to the reference species in the Genbank, Naegleria schusteri (AJ566626) and Naegleria laresi (AJ566630), respectively.