PCR-based diagnosis of Naegleria sp. infection in formalin-fixed and paraffin-embedded brain sections

We developed a real-time PCR which allowed the highly sensitive detection of Naegleria fowleri in histological brain tissue sections from experimentally infected mice. This genus-specific small-subunit (18S) rRNA gene-based PCR can complement conventional (immuno-) histology for the diagnosis of primary amoebic meningoencephalitis in paraffin-embedded brain necropsy specimens that had been fixed in formalin buffered with phosphate-buffered saline.

Organotypic slice cultures from rat brain tissue: a new approach for Naegleria fowleri CNS infection in vitro

The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

Species in the genus Naegleria are free-living amoebae of the soil and warm fresh water. Although around 30 species have been recognized, Naegleria fowleri is the only one that causes primary amoebic meningoencephalitis (PAM) in humans. PAM is an acute and fast progressing disease affecting the central nervous system. Most of the patients die within 1-2 weeks of exposure to the infectious water source. The fact that N. fowleri causes such fast progressing and highly lethal infections has opened many questions regarding the relevant pathogenicity factors of the amoeba. In order to investigate the pathogenesis of N. fowleri under defined experimental conditions, we developed a novel high- versus low-pathogenicity model for this pathogen. We showed that the composition of the axenic growth media influenced growth behaviour and morphology, as well as in vitro cytotoxicity and in vivo pathogenicity of N. fowleri. Trophozoites maintained in Nelson's medium were highly pathogenic for mice, demonstrated rapid in vitro proliferation, characteristic expression of surface membrane vesicles and a small cell diameter, and killed target mouse fibroblasts by both contact-dependent and -independent destruction. In contrast, N. fowleri cultured in PYNFH medium exhibited a low pathogenicity, slower growth, increased cell size and contact-dependent target cell destruction. However, cultivation of the amoeba in PYNFH medium supplemented with liver hydrolysate (LH) resulted in trophozoites that were highly pathogenic in mice, and demonstrated an intermediate proliferation rate in vitro, diminished cell diameter and contact-dependent target cell destruction. Thus, in this model, the presence of LH resulted in increased proliferation of trophozoites in vitro and enhanced pathogenicity of N. fowleri in mice. However, neither in vitro cytotoxicity mechanisms nor the presence of membrane vesicles on the surface correlated with the pathologic potential of the amoeba. This indicated that the pathogenicity of N. fowleri remains a complex interaction between as-yet-unidentified cellular mechanisms.

Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri.

BACKGROUND The free-living amoeba Naegleria fowleri is the causative agent of the rapidly progressing and typically fatal primary amoebic meningoencephalitis (PAM) in humans. Despite the devastating nature of this disease, which results in > 97% mortality, knowledge of the pathogenic mechanisms of the amoeba is incomplete. This work presents a comparative proteomic approach based on an experimental model in which the pathogenic potential of N. fowleri trophozoites is influenced by the compositions of different media. RESULTS As a scaffold for proteomic analysis, we sequenced the genome and transcriptome of N. fowleri. Since the sequence similarity of the recently published genome of Naegleria gruberi was far lower than the close taxonomic relationship of these species would suggest, a de novo sequencing approach was chosen. After excluding cell regulatory mechanisms originating from different media compositions, we identified 22 proteins with a potential role in the pathogenesis of PAM. Functional annotation of these proteins revealed, that the membrane is the major location where the amoeba exerts its pathogenic potential, possibly involving actin-dependent processes such as intracellular trafficking via vesicles. CONCLUSION This study describes for the first time the 30 Mb-genome and the transcriptome sequence of N. fowleri and provides the basis for the further definition of effective intervention strategies against the rare but highly fatal form of amoebic meningoencephalitis.

The ancient gene C12orf29 : an exploration of its role in the chordate body plan

The sheep (Ovis aries) is commonly used as a large animal model in skeletal research. Although the sheep genome has been sequenced there are still only a limited number of annotated mRNA sequences in public databases. A complementary DNA (cDNA) library was constructed to provide a generic resource for further exploration of genes that are actively expressed in bone cells in sheep. It was anticipated that the cDNA library would provide molecular tools for further research into the process of fracture repair and bone homeostasis, and add to the existing body of knowledge. One of the hallmarks of cDNA libraries has been the identification of novel genes and in this library the full open reading frame of the gene C12orf29 was cloned and characterised. This gene codes for a protein of unknown function with a molecular weight of 37 kDa. A literature search showed that no previous studies had been conducted into the biological role of C12orf29, except for some bioinformatics studies that suggested a possible link with cancer. Phylogenetic analyses revealed that C12orf29 had an ancient pedigree with a homologous gene found in some bacterial taxa. This implied that the gene was present in the last common eukaryotic ancestor, thought to have existed more than 2 billion years ago. This notion was further supported by the fact that the gene is found in taxa belonging to the two major eukaryotic branches, bikonts and unikonts. In the bikont supergroup a C12orf29-like gene was found in the single celled protist Naegleria gruberi, whereas in the unikont supergroup, encompassing the metazoa, the gene is universal to all chordate and, therefore, vertebrate species. It appears to have been lost to the majority of cnidaria and protostomes taxa; however, C12orf29-like genes have been found in the cnidarian freshwater hydra and the protostome Pacific oyster. The experimental data indicate that C12orf29 has a structural role in skeletal development and tissue homeostasis, whereas in silico analysis of the human C12orf29 promoter region suggests that its expression is potentially under the control of the NOTCH, WNT and TGF- developmental pathways, as well SOX9 and BAPX1; pathways that are all heavily involved in skeletogenesis. Taken together, this investigation provides strong evidence that C12orf29 has a very important role in the chordate body plan, in early skeletal development, cartilage homeostasis, and also a possible link with spina bifida in humans.

Non-Acanthamoeba amebic keratitis

Purpose. To describe the clinical presentation and outcome of two cases with presumed non-Acanthamoeba amebic keratitis. Methods. Case reports. Results. Both patients presented with typical symptoms and signs of Acanthamoeba keratitis. The patients' soft contact lenses, lens cases, open solutions, and conjunctival samples were cultured. Diagnosis of non- Acanthamoeba amebic keratitis was based on the presence of keratitis and amebic growth from patients' contact lenses of the affected eyes. Amebic culture from the contralateral contact lens was negative. Vahlkampfia cysts were identified in case 1, and Naegleria cysts in case 2. Topical treatment with polyhexamethylene biguanide and propamidine resolved the keratitis in case 1. Case 2 was lost to follow-up. Conclusion. Non-Acanthamoeba amebic keratitis was diagnosed in two patients based on the clinical presentation (resembling Acanthamoeba keratitis), culture of the contact lens, and response to antiamebic treatment (in one case). Keratitis associated with Naegleria contamination of contact lenses has not been previously reported.

Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant

The complex ecology of free-living amoebae (FLA) and their role in spreading pathogenic microorganisms through water systems have recently raised considerable interest. In this study, we investigated the presence of FLA and amoebae-resisting bacteria (ARB) at various stages of a drinking water plant fed with river water. We isolated various amoebal species from the river and from several points within the plant, mostly at early steps of water treatment. Echinamoeba- and Hartmannella-related amoebae were mainly recovered in the drinking water plant whereas Acanthamoeba- and Naegleria-related amoebae were recovered from the river water and the sand filtration units. Some FLA isolates were recovered immediately after the ozonation step, thus suggesting resistance of these microorganisms to this disinfection procedure. A bacterial isolate related to Mycobacterium mucogenicum was recovered from an Echinamoeba-related amoeba isolated from ozone-treated water. Various other ARB were recovered using co-culture with axenic Acanthamoeba castellanii, including mycobacteria, legionella, Chlamydia-like organisms and various proteobacteria. Noteworthy, a new Parachlamydia acanthamoebae strain was recovered from river water and from granular activated carbon (GAC) biofilm. As amoebae mainly multiply in sand and GAC filters, optimization of filter backwash procedures probably offers a possibility to better control these protists and the risk associated with their intracellular hosts

Amibes à potentiel pathogène dans les unités dentaires

Il a été bien documenté que les différentes canalisations des unités de soins dentaires contiennent un épais biofilm. Ce biofilm est constitué entre autres de bactéries, mais aussi d’amibes. Certaines amibes ont un potentiel pathogène et peuvent causer des infections graves. Deux cas d’infections amibiennes et possiblement reliées aux unités dentaires ont retenu notre attention et sont à l’origine du présent projet. L’identification morphologique des amibes afin de déterminer si elles présentent un potentiel pathogène ou non est une tâche ardue, même pour les protozoologistes chevronnés. Nous avons donc utilisé la réaction de polymérase en chaîne (PCR) pour identifier les amibes. Des nouvelles amorces ont été élaborées pour détecter les amibes des genres Acanthamoeba ainsi que Naegleria. Des échantillons d’eau et de terre ont été prélevés dans l’environnement, et des échantillons d’eau et de biofilm ont été prélevés dans les unités dentaires. Une partie de chaque échantillon a été mise en culture selon une méthode améliorée pour une identification morphologique, et l’autre partie a été soumise à un PCR direct. Des Acanthamoebae et/ou des Naegleriae ont été détectées dans 100% des échantillons, mais les espèces varient d’un échantillon à l’autre. Des amibes à potentiel pathogènes sont détectables dans les unités dentaires ainsi que dans l’environnement, et celles-ci pourraient représenter un risque pour la santé de certains individus.

Isolamento e identificação de amebas de vida livre potencialmente patogênicas em amostras de ambientes do Hospital de Clínicas de Porto Alegre - RS

Amebas de vida livre (AVL), tais como Acanthamoeba spp., Naegleria spp. e Balamuthia mandrillaris, são potenciais agentes de infecções humanas podendo ser encontradas no meio ambiente como solo, água fresca e ar atmosférico. No Brasil, de um modo geral, há poucos trabalhos relatando a importância do estudo desses patógenos em ambientes hospitalares. Assim este trabalho visou estudar a presença de Acanthamoeba spp. e Naegleria spp. na poeira e biofilmes de 15 ambientes diferentes (CTI, UTI pediátrica, Centro Cirúrgico, Centro Cirúrgico Ambulatorial, Emergência, Cozinha, Reservatórios de Azulejo e de Concreto, 06 Bebedouros e 01 Torneira) do Hospital de Clínicas de Porto Alegre, RS (HCPA). Coletas mensais de poeira e biofilmes foram realizadas com suabes passados aleatoriamente nos locais de coleta, de julho de 2004 e março de 2005, totalizando 135 amostras. Após sedimentação do material, o sedimento foi usado como inóculo em placas de Petri com ágar não nutriente 1,5%, previamente inoculadas com E. coli. As amostras foram incubadas durante 10 dias a 30°C. Das 135 amostras coletadas dos 15 ambientes do HCPA, 47 (35%) foram positivas para AVL, segundo critérios morfológicos de Page. Destas, 34% apresentaram características morfológicas próprias do gênero Acanthamoeba, sendo 03 desses isolados confirmados por PCR.

Isolamento de amebas de vida livre potencialmente patogênicas em poeira de hospitais

OBJETIVO: Observar a ocorrência de amebas de vida livre dos gêneros Acanthamoeba e Naegleria em amostras de poeira coletadas em hospitais. MÉTODOS: Foram coletadas 132 amostras de poeira em dois hospitais do município de Presidente Prudente, São Paulo. Os locais da coleta foram: Unidade de Terapia Intensiva, Centro Cirúrgico, Isolamento de Moléstias Infecciosas, Berçário, Emergência e Cozinha. As amostras foram semeadas em três meios de cultura: meio de ágar não nutriente com Escherichia coli, meio de ágar infusão de soja e microcultivo em meio de Pavlova modificado por Giazzi. As amebas isoladas foram identificadas segundo critérios morfológicos. RESULTADOS: O índice geral de positividade para amebas de vida livre, potencialmente patogênicas, dos gêneros Acanthamoeba e Naegleria, foi de 45,5%, sendo positivas 41,6% das amostras de poeira coletadas no hospital universitário e 50% no hospital estadual. Obtiveram-se 45,5% de positividade do gênero Acanthamoeba e 3,8% para amebas do gênero Naegleria. CONCLUSÕES: As amebas de vida livre, potencialmente patogênicas, estavam presentes em todos os ambientes estudados dos dois hospitais, sendo que as espécies do gênero Acanthamoeba foram as isoladas com maior freqüência.

Screening of Swiss hot spring resorts for potentially pathogenic free-living amoebae

Free-living amoebae (FLA) belonging to Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Sappinia pedata are known to cause infections in humans and animals leading to severe brain pathologies. Worldwide, warm aquatic environments have been found to be suitable habitats for pathogenic FLA. The present study reports on screening for potentially pathogenic FLA in four hot spring resorts in Switzerland. Water samples were taken from water filtration units and from the pools, respectively. Amoebae isolated from samples taken during, or before, the filtration process were demonstrated to be morphologically and phylogenetically related to Stenoamoeba sp., Hartmannella vermiformis, Echinamoeba exundans, and Acanthamoeba healyi. With regard to the swimming pools, FLA were isolated only in one resort, and the isolate was identified as non-pathogenic and as related to E. exundans. Further investigations showed that the isolates morphologically and phylogenetically related to A. healyi displayed a pronounced thermotolerance, and exhibited a marked in vitro cytotoxicity upon 5-day exposure to murine L929 fibroblasts. Experimental intranasal infection of Rag2-immunodeficient mice with these isolates led to severe brain pathologies, and viable trophozoites were isolated from the nasal mucosa, brain tissue, and lungs post mortem. In summary, isolates related to A. healyi were suggestive of being potentially pathogenic to immunocompromised persons. However, the presence of these isolates was limited to the filtration units, and an effective threat for health can therefore be excluded.

Screening Swiss water bodies for potentially pathogenic free-living amoebae

Free-ling amoebae (FLA) including Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris and Sappinia pedata, can cause opportunistic infections leading to severe brain pathologies. Human infections with pathogenic FLA have been increasingly documented in many countries. In Switzerland, thus far, the occurrence and distribution of potentially pathogenic FLA has not been investigated. Swiss water biotopes, including swimming pools, lakes, rivers and ponds, have now been screened for the presence of FLA, and assessment of their pathogenicity potential for a mammalian host has been undertaken. Thus, a total of 17 isolates were recovered by in vitro cultivation from these different aquatic sources. Characterization by sequence analysis of Acanthamoeba spp.-specific and 'FLA-specific PCR products amplified from 18s rDNA based on morphological traits, thermotolerance, and cytotoxicity towards murine fibroblasts yielded the following findings: Echinamoeba cf. exundans (3 isolates), Hartmannella spp. (3), Vannella spp. (4), Protacanthamoebica cf. bohemica (1), Acanthamoeba cf. castellanii (1) and Naegleria spp. (5). B. mandrillaris and N. fowleri did not range amongst these isolates. None of the isolates exhibited pronounced cytotoxicity and all failed to grow at 42 degrees C; therefore, they do not present any potential for CNS pathogenicity for humans.

Statnote 34 : the Kolmogorov-Smirnov (KS) test

The Kolmogorov-Smirnov (KS) test is a non-parametric test which can be used in two different circumstances. First, it can be used as an alternative to chi-square (?2) as a ‘goodness-of-fit’ test to compare whether a given ‘observed’ sample of observations conforms to an ‘expected’ distribution of results (KS, one-sample test). An example of the use of the one-sample test to determine whether a sample of observations was normally distributed was described previously. Second, it can be used as an alternative to the Mann-Whitney test to compare two independent samples of observations (KS, two-sample test). Hence, this statnote describes the use of the KS test with reference to two scenarios: (1) to compare the observed frequency (Fo) of soil samples containing cysts of the protozoan Naegleria collected each month for a year with an expected equal frequency (Fe) across months (one-sample test), and (2) to compare the abundance of bacteria on cloths and sponges sampled in a domestic kitchen environment (two-sample test).