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PubMed1988

An in vitro comparative study on the effect of amphotericin B, econazole, and 5-fluorocytosine on Naegleria fowleri, Naegleria australiensis, and Naegleria australiensis s.sp. italica.

We tested in vitro amphotericin B (AMP-B), econazole (ECO), and 5-fluorocytosine (5-FC) on pathogenic Naegleria fowleri (KUL strain), Naegleria australiensis s.sp. italica (AB-T-F3, original strain) to assess their sensitivity to chemotherapeutic compounds. Previous reports have shown the polyene antibiotic AMP-B to be the most active agent. It was, however, much more active on N. fowleri than on N. australiensis and N. australiensis s.sp italica. 5-FC and ECO gave rise to non appreciable effect at non-toxic corresponding dosages in vivo. The results of these in vitro tests are discussed.

Amphotericin B↗
PubMed2003

Detection of Naegleria spp. and Naegleria fowleri: a comparison of flagellation tests, ELISA and PCR.

To detect Naegleria spp, in particular Naegleria fowleri, the causative agent of human primary amoebic meningoencephalitis, a flagellation test (FT) is routinely used followed by a specific ELISA. A positive FT indicates the presence of Naegleria spp although some false negatives are likely to occur since parameters for enflagellation vary greatly. As negative FTs are not routinely screened any further for the presence of N. fowleri, this could result in an underestimation of the presence of this pathogen. Therefore, amoebae were further analysed using ELISA and standard PCR not only after a positive but also after a negative FT. In this study 39 cultures containing amoebae were tested with FT, ELISA and the two PCR assays with 11 positive for FT. These were submitted to ELISA and four confirmed as N. fowleri. PCR with the common primer-set on these 11 positive FTs revealed all as Naegleria spp. The specific PCR used on these cultures detected four positive for N. fowleri, corresponding totally with the ELISA results. The 28 negative flagellation tests were also submitted to ELISA and PCR. Of these, 11 were identified as Naegleria spp with common PCR and six as N. fowleri as well as with ELISA and the specific PCR. When the detection of Naegleria spp is based on intermediary processes, such as flagellation tests, false negatives are likely to occur leading to severe underestimations. This study has shown that amoebae taken from negative FTs can be identified as Naegleria spp and N. fowleri when using PCR and ELISA. The application of at least one of the specific N. fowleri tests is recommended for routine screening. The heterogeneous distribution of the false negative results between the different power plants suggested the presence of different genotypes.

Amebiasis↗
PubMed1983

Comparison of Naegleria fowleri and Naegleria gruberi cultivated in the same nutrient medium.

The human pathogenic amoeboflagellate Naegleria fowleri and the nonpathogenic species N. gruberi can be cultivated axenically but usually in different media. Naegleria fowleri 6088 has been adapted to grow in Balamuth H-4 medium, usually used to propagate N. gruberi nB81, and nB81 has been adapted to grow in supplemented Nelson's medium, usually used to propagate N. fowleri. N. gruberi nB81, grown in either medium, enflagellated 135 to 150 min after subculture to non-nutrient amoeba saline, whereas 6088 required 225 min. Naegleria gruberi nB81 grown in either medium was agglutinated by 100 micrograms concanavalin A/ml, whereas N. fowleri 6088 was not. Naegleria fowleri and N. gruberi grown in Nelson's medium became rounded to a greater extent upon chilling at 5 degrees C and remained rounded longer than Naegleria grown in Balamuth medium. The specificity of the surface antigens was an inherent characteristic of each species and not dependent upon the propagating medium, but Naegleria grown in Nelson's medium was agglutinated more reproducibly and more effectively by antiserum. N. gruberi was somewhat more resistant to acriflavine, actinomycin D, cycloheximide, or tetracycline than N. fowleri, regardless of the culture medium. Naegleria fowleri 6088 grown in Nelson's medium, however, was more resistant to actinomycin D, daunomycin, mithramycin, sulfamethoxazole, or tyrocidine than 6088 grown in Balamuth medium. There are limitations on the validity of comparisons of N. fowleri and N. gruberi based upon cultures grown in different media.

Agglutination Tests↗
PubMed2025

Detection and identification of Naegleria species along with Naegleria fowleri in the tap water samples.

Naegleria fowleri, the causative agent of Primary Amoebic Meningoencephalitis (PAM), is commonly found in warm freshwater environments and can enter the brain through nasal passages during activities like swimming or ablution. PAM has a high fatality rate, raising concerns about its global health impact. In Pakistan, particularly in Karachi, a significant number of cases have been reported, often with no history of recreational water exposure, but with regular ablution using tap water. This study analyzed the physicochemical parameters, abundance of total and fecal coliforms, and detected N. fowleri and other Naegleria species in tap water samples from Karachi using PCR with ITS- and Naegl-primers. Almost all samples exhibited high temperatures, low chlorine levels, and a high presence of coliforms. N. fowleri and other Naegleria species were detected in 11 out of 39 samples. Sequence analysis identified N. fowleri in tap water from the Golimar and Lyari areas of Karachi, while the other nine samples revealed different Naegleria species. This study suggests that the combination of high temperatures, insufficient chlorination, and the presence of coliforms may create favorable conditions for N. fowleri growth. However, these factors are not exclusive to the Golimar and Lyari areas, indicating that other environmental or infrastructural factors, not detailed in this study, may have contributed to the presence of N. fowleri in that specific location.

Naegleria fowleri↗
PubMed2001

The amoeba-to-flagellate transformation test is not reliable for the diagnosis of the genus Naegleria. Description of three new Naegleria spp.

Trophozoites of several isolates from one location in Australia have failed consistently to transform into flagellates, although they display all other characteristics of the genus Naegleria. When changing the standard transformation test, flagellates were produced. In phylogenetic trees derived from partial small subunit ribosomal DNA (SSUrDNA) sequences, one of these strains branches close to a cluster comprising N. clarki, N. australiensis, N. italica and N. jadini. It is proposed that these Australian isolates represent a new species, named N. fultoni (strain NG885). Failing to form flagellates since their isolation, even when different transformation procedures are used, are two Naegleria strains from Chile and Indonesia. In SSUrDNA-based phylogenetic trees the Chilean strain clusters with N. pussardi and the Indonesian strain clusters with N. galeacystis, but the degree of sequence difference from these described species (3.5% and 2.2%, respectively) is sufficient to propose that both of the strains represent new species, named N. chilensis (strain NG946) and N. indonesiensis (strain NG945), respectively. The close relationships between each of the new species and the Naegleria species with which they cluster in SSUrDNA-based trees were confirmed by ribosomal internal transcribed spacer region (ITS) sequence comparisons. In France, several non-flagellating N. fowleri strains were isolated from one location. ITS rDNA sequence comparisons indicated that they correspond to a 'type' of N. fowleri found in both Europe and the USA. A redefinition of the genus Naegleria is proposed as a consequence of these and previous findings.

Amebiasis↗
PubMed1987

Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoeba Naegleria lovaniensis and Naegleria gruberi.

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniesis, it is accompanied by lanosterol and parkeol, as well as by the 24,25-dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4 alpha-methylsterols which are present in similar amounts as those of 4,4-desmethylsterols (3-5 mg/g, dry weight). 4 alpha-Methylergosta-7,22-dienol was identified as a new compound. Ergosterol was the major 4,4-desmethylsterol, accompanied by small amounts of C27 and other C28 sterols. Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4 alpha-methyl-delta 7-sterols and the appearance of the unusual delta 6,8,22-ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the delta 8----delta 7 isomerase, the small amounts of delta 7-sterols formed being converted into ergosterol which is still present in fenpropimorph-exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1-14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3-3H]squalene epoxide was efficiently cyclized by a cell-free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non-photosynthetic phyla (animal and fungi) and parkeol are more likely dead-ends of this biosynthetic pathway. The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine-like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics.(ABSTRACT TRUNCATED AT 400 WORDS)

Amoeba↗
PubMed1993

Differentiation of Naegleria fowleri and other Naegleriae by polymerase chain reaction and hybridization methods.

In order to detect and identify Naegleria fowleri strains an assay based on the Polymerase Chain Reaction (PCR) was evaluated. The amplified DNA fragments were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot hybridization with an internal digoxigenin-labeled probe. A set of primers (B1B2) which flank a 678-bp region within a virulence-associated gene, allowed for the highly specific identification of N. fowleri, since Naegleriae (N. lovaniensis, N. australiensis, N. gruberi, N. andersoni and N. jadini) and other Protozoa did not react. These primers did not detect amplification products from various organisms: Gram-positive bacteria, Gram-negative bacteria, algae, yeasts and human DNA. Whereas a second set of primers (A1A2), which flank a different sequence, detected various Naegleriae and Acanthamoebae strains. After 40 amplification cycles, the limit of detection was a single cell (cyst or trophozoite). Thus, the PCR appears to be a rapid and powerful tool for identification and detection of N. fowleri.

Animals↗
PubMed1975

In vitro effects of amphotericin B on growth and ultrastructure of the amoeboflagellates Naegleria gruberi and Naegleria fowleri.

In vitro effects of the polyene antibiotic amphotericin B (AmB) on growth, viability, and ultrastructure of amoeboflagellates of the genus Naegleria were examined. The strains studied were the nonpathogenic Naegleria gruberi EG(B) and the Carter and TY strains of the pathogenic Naegleria fowleri. AmB was amoebicidal at all concentrations used (0.25, 0.50, and 1.0 mug/ml) when the drug was added to cultures in lag phase, as determined by viability testing, but was mainly inhibitory when added to log-phase cultures. The drug produced ultrastructural modifications at all concentrations (0.05 to 1.0 mug/ml). These changes included distortion of nuclear shape, increase in cytoplasmic membranes (both rough and smooth endoplasmic reticulum), decrease in number of food vacuoles, absence of pseudopod formation, mitochondrial abnormalities, increase in autophagic vacuoles, and blebbing of the plasma membrane. These alterations of amoebic ultrastructure became more pronounced with increased time in AmB and with increase in AmB concentration in the growth medium.

Amoeba↗
PubMed1992

Genotyping Naegleria spp. and Naegleria fowleri isolates by interrepeat polymerase chain reaction.

All six Naegleria species recognized to date were studied by interrepeat polymerase chain reaction (PCR). Priming at repeat sequences, which are known to be variable among eukaryotes, yielded electrophoretic DNA banding patterns that were specific for any single species. With a single PCR and simple gel electrophoresis, species determination could be performed in less than 1 day. Unambiguous discrimination between the pathogen N. fowleri and nonpathogenic Naegleria species appeared to be possible. Analysis of DNAs obtained from 20 separate isolates of N. fowleri revealed that geographic variation of the genetic fingerprints rarely occurs. All but 3 of 20 isolates of N. fowleri which were investigated showed identical banding patterns; for two isolates from New Zealand and one from Australia, a limited number of additional bands was detected, independent of the PCR primers used. These data corroborate previous findings on the genetic stability of pathogenic N. fowleri.

Animals↗
PubMed1993

Use of monoclonal antibodies to distinguish pathogenic Naegleria fowleri (cysts, trophozoites, or flagellate forms) from other Naegleria species.

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.

Animals↗
PubMed2006

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification.

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.

Actins↗
PubMed1982

Cytopathogenicity of Naegleria fowleri and Naegleria gruberi for established mammalian cell cultures.

Amebae of Naegleria fowleri and Naegleria gruberi were cytopathic for nine established mammalian cell cultures, including mouse and human fibroblasts, rabbit and monkey kidney cells, rat and mouse neuroblastoma cells, baby hamster kidney cells, and human epithelioma and carcinoma cells. Nine strains of N. fowleri were equally cytopathic for rodent neuroblastoma cells. As few as one ameba per million neuroblastoma cells destroyed the mammalian target cells after 9 days. The N. fowleri grew and destroyed rat neuroblastoma cells at 30 to 37 C whereas N. gruberi grew and destroyed the target cells at 25 to 30 C. Both N. fowleri and N. gruberi attached efficiently to the target cells at 30 to 37 C; N. gruberi but not N. fowleri attached efficiently at 25 C. Electron microscopic observations of mixed cultures of N. fowleri and neuroblastoma cells established that the amebae, after 12 hr, had ingested portions of the neuroblastoma target cells without causing cell lysis. Conversely, N. gruberi amebae, after attaching to target cells, disrupted the plasma membrane and cytoplasm of the target cells although the target cell nucleus remained intact. The amebae then ingested the target cell debris.

Amoeba↗
PubMed1987

Serology of Naegleria fowleri and Naegleria lovaniensis in a hospital survey.

An avidin-biotin horseradish peroxidase method was used to detect antibodies to Naegleria fowleri and N. lovaniensis in human serum samples. Antibodies were detected in 101 specimens from 115 hospital patients ranging in age from 15 to 98 years. Class-specific anti-immunoglobulins identified antibodies as IgG and IgM. IgG antibody titers to both species ranged from 1:20 to 1:640. Seven of 15 serum samples collected from newborn infants also demonstrated IgG antibodies to these organisms with a titer range of 1:20 to 1:80. The immunoperoxidase test and Western blot analysis of selected serum samples demonstrated a close similarity in serological results between N. fowleri and N. lovaniensis.

Adolescent↗
PubMed1991

Free-living amoebae in Egypt. 1. Naegleria gruberi and Naegleria fowleri.

Two Naegleria species were isolated and identified from various water sources in Lower and Upper Egypt. Identification was based on the morphology, nuclear division and the excystation and flagellation tests. The trophic, cystic and flagellate forms of N. gruberi are larger than those of N. fowleri and the cyst of the former species has one or more pores while that of the latter species has no pores and has an outer gelatinous layer. The size and the morphological characteristics of these two free-living amoebae from Egypt were in complete agreement with those previously described for amoebae of this same genus and species endemic to other geographical areas.

Animals↗
PubMed1983

Agglutination of Naegleria fowleri and Naegleria gruberi by antibodies in human serum.

The capability of serum samples from 423 human subjects to agglutinate rounded cells of Naegleria fowleri nN68 was assessed. Sera from the umbilical cords of seven infants failed to agglutinate N. fowleri cells. The median agglutination titer was 1:4 for sera from children through age 4 years, 1:8 for sera from juveniles 5 to 15 years of age, and 1:16 for sera from subjects 15 to 30 years old. The agglutination titers of sera from older adults decreased to a median of 1:8 for the 40- to 60-year-old age group and to 1:4 for the 60- to 90-year-old subjects. Serum samples from young adults agglutinated rounded cells of both N. fowleri and N. gruberi. The agglutination activity for N. fowleri was removed by absorption with N. fowleri but not with N. gruberi. Conversely, agglutination activity for N. gruberi was removed by absorption with N. gruberi but not with N. fowleri. The agglutinating activity for N. fowleri was immunoglobulin M. Serum samples from children displayed markedly disparate capabilities to agglutinate N. fowleri and N. gruberi. Only rounded cells of N. fowleri or N. gruberi were reliably agglutinated by human serum samples. Live or paraformaldehyde-killed cells could be used in the assay, but live N. gruberi cells returned to the amoeboid form, and these agglutinated poorly.

Adolescent↗
PubMed1985

Rapid identification of thermophilic Naegleria, including Naegleria fowleri using API ZYM system.

The suitability of the API ZYM system for identifying thermophilic Naegleria species, based on enzyme presence and activity, was investigated. Replicate testing on strains of N fowleri, N lovaniensis, and N australiensis cultured in a monoxenic and an axenic medium showed that the system could provide a rapid and reproducible means of identifying the species soon after primary isolation. No single enzyme was found specific for any one species, but considerable differences were found in the patterns of activity of acid phosphatase and leucine arylamidase. When these were compared the species could be differentiated. Use of the system in conjunction with a simple culture method is proposed as a readily available means of monitoring environmental and public bathing sites to prevent primary amoebic meningoencephalitis.

Acid Phosphatase↗