Vermamoeba vermiformis as etiological agent of a painful ulcer close to the eye.

In the present article, we report on the identification of Vermamoeba (Hartmannella) vermiformis as the etiological agent of a tissue infection close to the eye of a female patient. Laboratory examination revealed no involvement of any pathogenic bacteria or fungi in the tissue infection. V. vermiformis was identified by cultivation and morphology of trophozoites and cysts as well as phylogenetic analysis of nuclear 18S rDNA. The lesion improved in the course of 4 weeks by application of zinc paste.

Occurrence and molecular characterization of free-living amoeba species (Acanthamoeba, Hartmannella, and Saccamoeba limax) in various surface water resources of Iran.

This study was conducted to determine the presence and molecular identity of Acanthamoeba species in the surface water resources of four provinces in Iran, namely Guilan, Mazandaran (North of Iran), Alborz, and Tehran (capital city), using culture- and molecular-based methods. During March to November 2014, 49 surface water samples were collected from environmental water sources-the distinct surface waters of Guilan, Mazandaran, Alborz, and Tehran provinces, in Iran. For the isolation of Acanthamoeba species, approximately 500 ml of the water samples were filtered through a cellulose nitrate membrane with a pore size of 0.45 µ. The filter was transferred onto non-nutrient agar plates seeded with Gram-negative bacteria (Escherichia coli) as a food source. The presence of Acanthamoeba was confirmed by the genus-specific primer pair JDP1 and 2, and/or NA primers were used to identify Acanthamoeba and certain other free-living amoebae. In total, 38 out of 49 samples were positive by culture and/or PCR for Acanthamoeba and other free-living amoebae from all three provinces. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba (16 isolates belonged to T4 and 2 isolates belonged to T5), Hartmannella vermiformis (3/24), and Saccamoeba limax (2/24). The T4 and T5 genotypes were detected in Guilan and Mazandaran provinces. Two isolates from Guilan and Tehran provinces belonged to S. limax, and H. vermiformis was detected in Guilan province. The results of this study highlight the need to pay more attention to free-living amoebae, as human activity was observed in all of the localities from which these samples were taken. These surface waters can be potential sources for the distribution and transmission of pathogenic Acanthamoeba in the study areas, and free-living amoebas (FLA) (particularly the Acanthamoeba species) can serve as hosts for and vehicles of various microorganisms.

Thermophilic potentially pathogenic amoebae isolated from natural water bodies in Poland and their molecular characterization.

The free-living amoebae (FLA) may live in the environment and also within other organisms as parasites and then they are called amphizoic. They are potentially pathogenic for humans and animals and are found in water that is a source of infection. The aim of this study was molecular detection and identification of these FLA in natural water bodies in North-Western Poland to evaluate the risk of the pathogenic amoebae infections. We examined surface water samples collected from 50 sites and first, the tolerance thermic test was performed in order to select thermophilic, potentially pathogenic strains. For molecular identification of FLA, regions of 18S rDNA, 16S rDNA and intergenic spacers were amplified. Acanthamoeba T4 and T16 genotypes of 18S rDNA gene and 18S rDNA of H. vermiformis were detected. We identified two variants of Acanthamoeba T4 genotype, two variants of Acanthamoeba T16 genotype and one variant of H. vermiformis. Identification of the T16 genotype and H. vermiformis in water was for the first time in Poland. Additionally, we made attempts to adapt the RLB method for detection and differentiation of FLA species and strains. PCR seems to be more sensitive than RLB hybridization, though.

The prevalence, isolation and morphotyping of potentially pathogenic free-living amoebae from tap water and environmental water sources in Sivas.

OBJECTIVE: To our knowledge, there is no study dealing with the prevalence of free-living amoebas (FLA) in water sources in Turkey, previous studies were mostly case presentations. The aim of the present study was to investigate the prevalence of FLA from tap water and natural water sources in different parts of the city. METHODS: In the study, 250 samples were collected from the city centre, districts and villages. Two litres of water was collected from each source and filtered through a vacuum filtration system. The filter papers were washed in "Page's Amoeba Saline (PAS)" solution and incubated overnight. Filter papers were removed from the tubes and centrifuged; the final pellet was inoculated on non-nutrient agar (NNA) plates. The growth rate of FLA was checked after three days of inoculation and the flagellation test was performed to determine the presence of Naegleria spp. Heat tolerance of isolated strains was checked at 37, 42 and 52°C for the presence of pathogenic Acanthamoeba species. The cyst and trophozoite morphology of amoebas were examined under a light microscope and the genera was identified according to morphotyping keys. RESULTS: FLA were found in 75 (30.0%) of examined water samples. Eleven (4.4%) were identified as Acanthamoeba spp., 25 (10.0%) as Naegleria spp. and 39 (15.6%) as Hartmannella spp. after microscopic examination. CONCLUSION: Our study revealed that FLA are common inhabitants of household water as they are in the environment, so their own potential risks as well as transferring bacteria as other pathogens is important for human health.

Isolation and identification of Legionella and their host amoebae from weak alkaline carbonate spring water using a culture method combined with PCR.

Legionella were detected with the direct DNA extraction method, Legionella culture method, and free-living amoebae (FLA) culture method from weak alkaline carbonate spring water in Taiwan. Moreover, we also investigated the existence of Acanthamoeba, Hartmannella, and Naegleria, ubiquitous FLA in aquatic environments, to identify the correlations between existing Legionella. This study reports detecting Legionella in 15 of the 51 weak alkaline carbonate spring water samples (29.4%). This work also found five of the 51 samples (9.8%) analyzed by the direct DNA extraction method, three of the 51 (5.9%) samples analyzed by the Legionella culture method, and 11 of the 51 samples (21.6%) evaluated using the FLA culture method to be positive for Legionella. The most frequently identified Legionella species was the Legionella-like amoebal pathogen (n=5), followed by unidentified Legionella spp. (n=4), and Legionella pneumophila (n=4), Legionella fairfieldensis (n=3), and then Legionella rubrilucens (n=2). Legionella waltersii was detected once. The occurrence of Acanthamoeba, Hartmannella, and Naegleria were 5.9% (3/51), 52.9% (27/51), and 5.9% (3/51), respectively. All Hartmannella isolates were identified as Hartmannella vermiformis, and Naegleria isolates were all identified as Naegleria australiensis. The three Acanthamoeba isolates were identified as one Acanthamoeba polyphaga and two Acanthamoeba jacobsi. H. vermiformis (40.7%) were Legionella hosts, including all of the amoebae-resistant Legionella detected in the present study. Therefore, the important correlations between Legionella and H. vermiformis require further clarification. The combined results of this survey confirm that Legionella and FLA are ubiquitous in weak alkaline carbonate spring water in Taiwan.

Potentially pathogenic free-living amoebae isolated from hospital wards with immunodeficient patients in Tehran, Iran.

This study investigated the occurrence of free-living amoebae (FLA) in immunodeficiency wards of hospitals in Tehran, Iran. A total of 70 dust and biofilm samples from wards serving transplant, pediatric (malignancies), HIV, leukemia and oncology patients of five university hospitals were collected and examined for the presence of FLA using culturing and molecular approaches. Based on the morphology of the amoebae in plate cultures, primer sets were applied for molecular identification of Acanthamoeba, vahlkampfiid amoebae and Hartmannella. Out of 70 samples, 37 (52.9%) were positive for FLA. Acanthamoeba belonged to the T4 genotype was the most prevalent isolate. Presence of the T4 genotype on medical instruments, including an oxygen mask in an isolation room of an immunodeficiency pediatric ward, should be of concern for health authorities. Acanthamoeba T5 genotypes, Hartmannella vermiformis, and Vahlkampfia avara were also present. These results highlight a clear need for greater attention to improved disinfection, especially where susceptible patients, such as those who are immune-suppressed, are served. To our knowledge, this is the first report of these FLA in immunodeficiency wards in Iran, and also the first to identify Acanthamoeba T5, Hartmannella, and Vahlkampfia in moist habitats, such as biofilms, in this country.

Survey of pathogenic free-living amoebae and Legionella spp. in mud spring recreation area.

Acanthamoeba, Hartmannella, and Naegleria are free-living amoebae, ubiquitous in aquatic environments. Several species within these genera are recognized as potential human pathogens. These free-living amoebae may facilitate the proliferation of their parasitical bacteria, such as Legionella. In this study, we identified Acanthamoeba, Hartmannella, Naegleria, and Legionella using various analytical procedures and investigated their occurrence at a mud spring recreation area in Taiwan. We investigated factors potentially associated with the prevalence of the pathogens, including various water types, and physical and microbiological water quality parameters. Spring water was collected from 34 sites and Acanthamoeba, Hartmannella, Naegleria, and Legionella were detected in 8.8%, 35.3%, 14.7%, and 47.1%, respectively. The identified species of Acanthamoeba included Acanthamoeba castellanii and Acanthamoeba polyphaga. Nearly all the Hartmannella isolates are identified as Hartmannella vermiformis. The Naegleria species included Naegleria australiensis and its sister groups, and two other isolates referred to a new clade of Naegleria genotypes. The Legionella species identified included unnamed Legionella genotypes, Legionella pneumophila serotype 6, uncultured Legionella spp., Legionella lytica, Legionella drancourtii, and Legionella waltersii. Significant differences (Mann-Whitney U test, P<0.05) were observed between the presence/absence of Hartmannella and total coliforms, between the presence/absence of Naegleria and heterotrophic plate counts, and between the presence/absence of Legionella and heterotrophic plate counts. This survey confirms that pathogenic free-living amoebae and Legionella are prevalent in this Taiwanese mud spring recreation area. The presence of pathogens should be considered a potential health threat when associated with human activities in spring water.

Quantitative detection and differentiation of free-living amoeba species using SYBR green-based real-time PCR melting curve analysis.

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.

[Infections caused by free-living amebas. Historical commentaries, taxonomy and nomenclature, protozoology and clinicopathologic features]. / Infecciones por amebas de vida libre. Comentarios históricos, taxonomía y nomenclatura, protozoología y cuadros anátomo-clínicos.

Infections caused by free-living amebae constitute one of emergent opportunistic infections with greatest medical interest. Although infrequently, they have been described in almost all world, its diagnosis depends on a high index of suspicion, especially in morpho-pathologic and laboratory studies. Exciting historical features of infections due to free-living amebae, its taxonomy and the present nomenclature are briefly reviewed. An analysis of the protozoology of the most frequent agents is done and, based on the author's own experience and the published one, already established anatomo-clinical entities are described: the primary amebic meningoencephalitis, granulomatous amebic encephalitis, Acanthamoeba keratitis, cutaneous acanthamoebiasis, disseminated infection and other rare isolated locations.

Fish-isolated strains of Hartmannella vermiformis page, 1967: morphology, phylogeny and molecular diagnosis of the species in tissue lesions.

Based on morphological and molecular characterisation, four amoeba strains isolated from organs of freshwater fish were identified as Hartmannella vermiformis Page, 1967. Small subunit rRNA gene sequences of these strains expand the set of corresponding complete and almost complete sequences of this species to twelve. A new species-specific oligonucleotide probe inferred from recently available SSU rRNA gene sequences was designed and successfully tested in tissue lesions produced by one strain of H. vermiformis in experimentally infected fish.

The identification of free-living environmental isolates of amoebae from Bulgaria.

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named "6" (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)--all exhibiting a highly efficient proliferation in axenic cultures--were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.

Hartmannella vermiformis isolated from the cerebrospinal fluid of a young male patient with meningoencephalitis and bronchopneumonia.

Meningoencephalitis and bronchopneumonia were documented in a patient from Peubla, Mexico. The patient began with symptoms and signs of a common flu and 12 days after the onset of his disease he was admitted to the hospital presenting symptoms and signs of meningoencephalitis. The clinical course evolved into an endocraneal hypertension syndrome with bronchopneumonia, coma and death. Wide-spectrum antibiotics, immunosuppressive and anti-tuberculosis therapy were unsuccessfully administered. Important antecedents were degree I malnutrition and repeated contact with polluted water. Post-mortem autopsy was not performed. Gram-positive cocci were isolated from the spinal fluid 2 days after admission, and then active amebae were isolated from three different samples of the spinal fluid at days 16, 18 and 19 after admission. Such samples were concentrated and inoculated onto specific culture media. Identification of amebae was based on their morphology and biochemistry. All amebae were Hartmannella vermiformis. Amebae were apparently not the cause of the disease and might be considered as an opportunistic colonizer which may have caused the evolution of the disease to become worse.

Primary amoebic meningoencephalitis.

As a serendipitous by-product of polio virus research, a highly fatal amoebic meningoencephalitis was recognized in animals. The causative microorganisms, contaminants of the viral cultures, were identified as small soil amoebae. These organisms, previously considered non-pathogenic, are prevalent throughout the world. Based on animal studies, the original investigators suggested the possibility of a similar disease in humans. Seven years later, human cases of amoebic meningoencephalitis were reported from widely separated areas of the world. Since 1965, a total of 79 cases have been reported. The literature of primary amoebic meningoencephalitis is presented. The history of the discovery and elucidation of this disease is reviewed. The 79 cases reported in the world literature are divided into two groups, those diagnosed retrospectively after reviewing previous deaths from meningoencephalitis, and those diagnosed at the time of the illness. The classification, morphology, pathogenicity, virulence and distribution of pathogenic soil amoebae are reviewed. The presenting clinical findings, diagnostic procedures, pathology, and management of this recently recognized, highly fatal, human disease is presented along with a report of a new case. Otolaryngologists should become familiar with this serious disorder with a transnasal portal of entry.

Electrophoretic characterization of small free-living Amoebae.

Disc electrophoresis was employed in an attempt to determine its usefulness as a means of differentiating and classifying some free-living species of amoebae. The Rp values and the percentage of similarity of the protein bands were used as taxonomic criteria. Some species of the genera Hartmanella and Naegleria were examined. The protein patterns of all species studied were highly reproducible. Four strains of Hartmannella castellanii showed an absolute identity to the original H. castellanii strain. One strain isolated by Neff and H. rhysodes were similar to all the other strains although their protein patten were not completely identical. On the other hand, H. culbertsoni, H. astronyxis and H. lammellipodia were different from all the other examined species of Hartmannella. Pathogenic N. fowleri-strains were identical to each other and were different in their protein patterns from non-pathogenic N. gruberi strain. The similarities and differences of protein bands of strains of ameobae and their correlations with other attributes were discussed. The results have shown that the disc electrophoresis appears useful in differentiating between species of Hartmannella and Naegleria.