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.     

Genetic Variations in the Internal Transcribed Spacer and Mitochondrial Small Subunit rRNA Gene of Naegleria spp.

ABSTRACT: Naegleria spp. are widely distributed free-living amebas, but one species in the genus, N. fowleri , causes acute fulminant primary amebic meningoencephalitis in humans and other animals. Thus, it is important to differentiate N. fowleri from the rest in the genus of Naegleria , and to develop tools for the detection of intra-specific genetic variations. In this study, one isolate each of N. australiensis, N. gruberi, N. jadini , and N. lovaniensis and 22 isolates of N. fowleri were characterized at the internal transcribed spacers (ITS) and mitochondrial small subunit rRNA (mtSSU rRNA) gene. The mtSSU rRNA primers designed amplified DNA of all isolates, with distinct sequences obtained from all species examined. In contrast, the ITS primers only amplified DNA from N. lovaniensis and N. fowleri , with minor sequence differences between the two. Three genotypes of N. fowleri were found among the isolates analyzed in both the mtSSU rRNA gene and ITS. The extent of sequence variation was greater in the mtSSU rRNA gene, but the ITS had the advantage of length polymorphism. These data should be useful in the development of molecular tools for rapid species differentiation and genotyping of Naegleria spp.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

Identification and epidemiological typing of Naegleria fowleri with DNA probes.

Naegleria fowleri is a small free-living amoeboflagellate found in warm water habitats worldwide. The organism is pathogenic to humans, causing fatal primary amoebic meningoencephalitis. When monitoring the environment for the presence of N. fowleri, it is important to reliably differentiate the organism from other closely related but nonpathogenic species. To this end, we have developed species-specific DNA probes for use in the rapid identification of N. fowleri from the environment. Samples were taken from the thermal springs in Bath, England, and cultured for amoebae. Of 84 isolates of thermophilic Naegleria spp., 10 were identified as N. fowleri by probe hybridization. The identity of these isolates was subsequently confirmed by their specific whole-cell DNA restriction fragment length polymorphisms (RFLPs). One DNA clone was found to contain a repeated element that detected chromosomal RFLPs that were not directly visible on agarose gels. This enabled the further differentiation of strains within geographically defined whole-cell DNA RFLP groups. N. fowleri DNA probes represent a specific and potentially rapid method for the identification of the organism soon after primary isolation from the environment.     

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.     

The immune response to Naegleria fowleri amebae and pathogenesis of infection

The genus Naegleria is comprised of a group of free-living ameboflagellates found in diverse habitats worldwide. Over 30 species have been isolated from soil and water but only Naegleria fowleri (N. fowleri) has been associated with human disease. Naegleria fowleri causes primary amebic meningoencephalitis (PAM), a fatal disease of the central nervous system. The pathogenesis of PAM and the role of host immunity to N. fowleri are poorly understood. Strategies for combating infection are limited because disease progression is rapid and N. fowleri has developed strategies to evade the immune system. The medical significance of these free-living ameboflagellates should not be underestimated, not only because they are agents of human disease, but also because they can serve as reservoirs of pathogenic bacteria.     

Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.     

Biochemical identification and phylogenetic relationships in free-living amoebas of the genus Naegleria

Using isoelectric focusing, the zymograms of 23 pathogenic and nonpathogenic Naegleria strains were studied for the activity of 16 enzymes. Certain enzymes (lactate dehydrogenase, L-threonine dehydrogenase, superoxide dismutase, acid phosphatase, malic enzyme, and leucine aminopeptidase) proved particularly useful from a practical point of view as they allow easy and reliable identification of pathogenic N. fowleri and N. australiensis as well as nonpathogenic N. lovaniensis strains. Genetic interpretation of these zymograms gave estimates of genetic distances that largely confirmed the taxonomic position of the Naegleria species. In addition, the genetic data suggest that there are two main phylogenetic groups in the genus Naegleria.     

Molecular definition and the ubiquity of species in the genus Naegleria

To investigate the variability within species of the genus Naegleria, the ITS1,5.8S and ITS2 rDNA were sequenced of several strains of N. lovaniensis and its Western Australian variants, N. australiensis, N. fowleri, N. andersoni, N. jamiesoni, N. tihangensis, N. pringsheimi, N. pagei, N. gruberi sensu lato and a Naegleria lineage that lost a group I intron from the SSUrDNA twintron. As a result, it is possible to define a molecular species within the Naegleria genus. In addition, one strain of each different allozyme cluster was sequenced to investigate whether they belong to described species or should be treated as distinct new species. This leads to the proposal of eleven new species. The sequencing results from those Naegleria spp. of which several strains are available indicate that these species are ubiquitous. The only exception might be the species represented by the WA variants. However, there are still many Naegleria spp. for which only one strain has been isolated, hence, it is important that the search for more isolates should be continued worldwide.     

Identification of Naegleria fowleri and other Naegleria spp. (free-living amoebae) using cellulose acetate membrane electrophoresis of glucose phosphate isomerase

Abstract A simple isoenzyme cellulose acetate membrane electrophoresis method with respect to glucose phosphate isomerase (GPI) was developed for the differentiation of the human pathogenic free-living amoeba Naegleria fowleri from other Naegleria spp. A single GPI band was detected in all the species tested, the relative mobility of which could be used to identify N. fowleri . Of the other Naegleria spp., only N. italica and N. jadini shared a common GPI mobility. No intraspecies variation in GPI profile was detected, regardless of whether the strains were cultured in monoxenic or axenic media. The technique is proposed as a useful means of identifying N. fowleri soon after isolation from the environment.     

Restriction endonuclease analysis of mitochondrial DNA as an aid in the taxonomy of Naegleria and Vahlkampfia

Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.     

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.     

Application of flow cytometry to studies of pathogenic free-living amoebae

Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleria fowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading.     

Occurrence of Naegleria and Acanthamoeba in aquaria

Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi.     

Thermal ecology of Naegleria fowleri from a power plant cooling reservoir

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 significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.     

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

Abstract 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, algae, y, 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 .     

Occurrence of Naegleria and Acanthamoeba in aquaria.

Samples from 24 aquaria were incubated at 28, 37, and 45 degrees C for the isolation of Naegleria and Acanthamoeba. Naegleria was the predominant genus (60.9%), whereas Acanthamoeba represented 15.5% of the isolates. No pathogenic N. fowleri was identified, although a high number of strains were closely related to this species. One isolate (Aq/9/1/45D) was compared with an aquarium isolate (PPMFB-6) from Australia. The Belgian isolate was found to be more related to N. fowleri, whereas the Australian isolate was closer to N. gruberi.     

Thermal ecology of Naegleria fowleri from a power plant cooling reservoir.

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 significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.     

Development of a PCR for identification of Naegleria fowleri from the environment.

A species-specific PCR for the identification of Naegleria fowleri was developed. In sensitivity studies, 10 trophozoites or cysts and 1 trophozoite or cyst could be detected after 35 and 45 cycles, respectively. In conjunction with a rapid DNA isolation method, this PCR was used to identify N. fowleri directly from primary cultures of environmental samples.