Saline-Tolerant Pathogenic Acanthamoeba spp. Isolated from a Geothermal Power Plant.

Few studies have been conducted in the cooling systems of power plants; they have focused on Naegleria fowleri, leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic Acanthamoeba in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of Acanthamoeba was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 104 to 8.7 × 104 µS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. Acanthamoeba was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of Acanthamoeba culbertsoni killed the mice, having a mortality of 40 to 100%. Acanthamoeba genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.

Naegleria australiensis isolated from a wastewater treatment station in Santiago Island, Cape Verde

Despite the Naegleria genus being isolated from different natural environments such as water, soil, and air, not all Naegleria species are capable of causing infections in humans, and they are capable of completing their life cycle in environmental niches. However, the presence of this genus may suggest the existence of one of the highly pathogenic free-living amoeba (FLA) species: Naegleria fowleri or the brain-eating amoeba. This facultative parasitic protozoon represents a risk to public health, mainly related to domestic and agricultural waters. In this research, our main objective was to determine the existence of pathogenic protozoa in the Santa Cruz wastewater treatment plant, Santiago Island. Using 5 L of water we confirmed the presence of potentially pathogenic Naegleria australiensis, being the first report on Naegleria species in Cape Verde. This fact demonstrates the low efficiency in the treatment of wastewater and, consequently, a potential threat to public health. Nevertheless, more studies will be needed for the prevention and control of possible infections in this Macaronesian country.

A history of over 40 years of potentially pathogenic free-living amoeba studies in Brazil - a systematic review.

Free-living amoeba (FLA) group includes the potentially pathogenic genera Acanthamoeba, Naegleria, Balamuthia, Sappinia, and Vermamoeba, causative agents of human infections (encephalitis, keratitis, and disseminated diseases). In Brazil, the first report on pathogenic FLA was published in the 70s and showed meningoencephalitis caused by Naegleria spp. FLA studies are emerging, but no literature review is available to investigate this trend in Brazil critically. Thus, the present work aims to integrate and discuss these data. Scopus, PubMed, and Web of Science were searched, retrieving studies from 1974 to 2020. The screening process resulted in 178 papers, which were clustered into core and auxiliary classes and sorted into five categories: wet-bench studies, dry-bench studies, clinical reports, environmental identifications, and literature reviews. The papers dating from the last ten years account for 75% (134/178) of the total publications, indicating the FLA topic has gained Brazilian interest. Moreover, 81% (144/178) address Acanthamoeba-related matter, revealing this genus as the most prevalent in all categories. Brazil's Southeast, South, and Midwest geographic regions accounted for 96% (171/178) of the publications studied in the present work. To the best of our knowledge, this review is the pioneer in summarising the FLA research history in Brazil.

A history of over 40 years of potentially pathogenic free-living amoeba studies in Brazil - a systematic review

Free-living amoeba (FLA) group includes the potentially pathogenic genera Acanthamoeba, Naegleria, Balamuthia, Sappinia, and Vermamoeba, causative agents of human infections (encephalitis, keratitis, and disseminated diseases). In Brazil, the first report on pathogenic FLA was published in the 70s and showed meningoencephalitis caused by Naegleria spp. FLA studies are emerging, but no literature review is available to investigate this trend in Brazil critically. Thus, the present work aims to integrate and discuss these data. Scopus, PubMed, and Web of Science were searched, retrieving studies from 1974 to 2020. The screening process resulted in 178 papers, which were clustered into core and auxiliary classes and sorted into five categories: wet-bench studies, dry-bench studies, clinical reports, environmental identifications, and literature reviews. The papers dating from the last ten years account for 75% (134/178) of the total publications, indicating the FLA topic has gained Brazilian interest. Moreover, 81% (144/178) address Acanthamoeba-related matter, revealing this genus as the most prevalent in all categories. Brazil's Southeast, South, and Midwest geographic regions accounted for 96% (171/178) of the publications studied in the present work. To the best of our knowledge, this review is the pioneer in summarising the FLA research history in Brazil.

Isolation and Identification of Naegleria Species in Irrigation Channels for Recreational Use in Mexicali Valley, Mexico.

Members of the genus Naegleria are free-living amoebae that are widely distributed in water and soil environments. Moreover, Naegleria fowleri is a pathogenic amoeba species that causes a fatal disease in the central nervous system known as primary amoebic meningoencephalitis (PAM) in humans. Since most reported infections due to N. fowleri are reported in recreational waters worldwide, this study was aimed to describe the presence of these amoebic genus in Mexicali Valley irrigation channels of recreational use. A total of nine water samples were collected and processed by triplicate, in nine different sites of the Valley. After filtering and culturing the samples, plates were examined, and the observed amoebae were morphologically identified at the genus level. In addition, the pathogenicity of these amoebic isolates was checked, and molecular characterization was performed by PCR/sequencing. The results revealed the presence of Naegleria spp. in all the channels sampled. Finally, molecular identification confirmed the presence of five different species of Naegleria: N. fowleri, N. australiensis, N. gruberi, N. clarki and N. pagei. The presence of these protists, particularly N. fowleri, should be considered as a potential human health risk in the region.

Isolation of Naegleria spp. from a Brazilian Water Source.

The genus Naegleria, of the free-living amoeba (FLA) group, has been investigated mainly due to its human health impact, resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the "brain-eating amoeba," is the most studied Naegleria species because it causes primary amoebic meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation, we present research on identifying Naegleria spp. in the Monjolinho River as a model study. The river is a public Brazilian freshwater source that crosses the city of São Carlos, in São Paulo state, Brazil. Five distinct sampling sites were examined through limnological features, trophozoites culturing, and PCR against internal transcribed spacer (ITS) regions and 5.8S rRNA sequences. The results identified N. philippinensis, N. canariensisi, N. australiensis, N. gruberi, N. dobsoni sequences, as well as a Hartmannella sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our results stress the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil.

Evaluation of the sensitivity to chlorhexidine, voriconazole and itraconazole of T4 genotype Acanthamoeba isolated from Mexico.

Free-living amoebae of the genus Acanthamoeba are the etiological agents of cutaneous lesions, granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK), which are chronic infections with poor prognosis if not diagnosed promptly. Currently, there is no optimal therapeutic scheme to eradicate the pathologies these protozoa cause. In this study we report the morphological and molecular identification of three species of the genus Acanthamoeba, belonging to T4 group; A. polyphaga isolated from the corneal ulcer of a patient sample of AK case; A. castellanii isolated from the contact lens of an AK patient and A. palestinensis obtained from a soil sample. The in vitro activity of chlorhexidine, itraconazole and voriconazole drugs against trophic stage was also evaluated through a colorimetric assay based on the oxidation-reduction of alamar blue. The strains in the study were sensitive to the evaluated drugs; although when determining the 50% inhibitory concentration (IC50) statistically significant differences were observed. A. castellanii showed to be highly sensitive to voriconazole (0.66 ±â€¯0.13 µM) but the least sensitive to chlorhexidine and itraconazole (8.61 ±â€¯1.63 and 20.14 ±â€¯4.93 µM, respectively), A. palestinensis showed the highest sensitivity to itraconazole (0.502 ±â€¯0.11 µM) and A. polyphaga expressed moderate sensitivity to chlorhexidine and itraconazole and lower sensitivity to voriconazole (10.10 ±â€¯2.21 µM). These results showed that species of the genus Acanthamoeba express different sensitivity to the tested drugs, which could explain the problems surrounding the establishment of a treatment of choice in the infections caused by these amoebae. We consider that although chlorhexidine and itraconazole show good activity on these amoebae and have been used in cases of AK in Mexico with acceptable results, voriconazole should be considered as the first therapeutic option of future Acanthamoeba infections that will be diagnosed in our country.

Acanthamoeba keratitis in Mexico: Report of a clinical case and importance of sensitivity assays for a better outcome.

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection. The early symptoms include redness, pain, photophobia and intense tearing. Chronic infection usually progresses to stromal inflammation, ring ulcers, corneal opacification and hypopyon. Here we document an AK case in a high myopic 38-year-old woman from Mexico City, with a history of wearing contact lenses while swimming. Corneal scrapes cultures were positive only for amoebae, consequently a treatment including netilmicin 0.3% and oral itraconazole 100 mg/12 h was prescribed. The infection was resolved after 8 months, leaving a slight leucoma outside the visual axis, with a visual acuity of 20/150. In the laboratory, the amoebic isolate was axenized in PYG medium, with an optimal growth at 30 °C, and was identified morphologically as Acanthamoeba polyphaga according to the taxonomic criteria of Page (1988) and placed in the T4 group by genotyping. The virulence of this strain (40%) was determined by intranasal inoculation of 1 × 106/20 µl trophozoites in BALB/c mice recovering from brain, proving their invasion ability and by the interaction with monolayers of epithelial cells of the established MDCK line of canine kidney origin (1:2 ratio of interaction), at 1, 3, 6, 8 and 24 h; trophozoites migrated to cell junctions inducing few lytic zones. In addition to the biological characterization, in vitro drug sensitivity tests were performed using chlorhexidine, itraconazole, netilmicin and voriconazole. Results revealed that voriconazole was the most effective compound. A. polyphaga remains as one of the most frequently isolated species producing AK. The treatment of AK case using netilmicin and oral itraconazole solved the disease, but the healing process was wide-ranging (8 months). The use of voriconazole and chlorhexidine may be an alternative treatment of future AK cases in Mexico.

Anti-Acanthamoeba Activity of Brominated Sesquiterpenes from Laurencia johnstonii.

Focused on our interest to develop novel antiparasistic agents, the present study was aimed to evaluate the biological activity of an extract of Laurencia johnstonii collected in Baja California Sur, Mexico, against an Acantamoeba castellanii Neff strain. Bioassay-guided fractionation allowed us to identify the amoebicidal diastereoisomers α-bromocuparane (4) and α-isobromocuparane (5). Furthermore, bromination of the inactive laurinterol (1) and isolaurinterol (2) yielded four halogenated derivatives, (6)⁻(9), which improved the activity of the natural sesquiterpenes. Among them, the most active compound was 3α-bromojohnstane (7), a sesquiterpene derivative which possesses a novel carbon skeleton johnstane.

Genotyping of clinical isolates of Acanthamoeba genus in Venezuela.

Free-living amoebae of Acanthamoeba genus are opportunistic pathogens distributed worldwide. Strains included in this genus are causative agents of a fatal encephalitis and a sight-threating keratitis in humans and other animals. In this study, 550 clinical samples which were collected between 1984 and 2014 from different patients with suspected infections due to Acanthamoeba were initially screened for the presence of this amoebic genus at the Laboratorio de Amibiasis-Escuela de Bioanálisis at the Universidad Central de Venezuela. Samples were cultured in 2% Non-Nutrient agar plates seeded with a layer of heat killed Escherichia coli. From the 550 clinical samples included in this study, 18 of them were positive for Acanthamoeba genus after culture identification. Moreover, positive samples were confirmed after amplification of the Diagnostic Fragment 3 (DF3) of the Acanthamoeba18S rDNA genus and sequencing was carried out in order to genotype the isolated strains of Acanthamoeba. Furthermore, the pathogenic potential of the strains was checked by performing thermotolerance and osmotolerance assays. Sequencing of the DF3 region resulted in the identification of genotype T4 in all the isolated strains. Moreover, most isolates were thermotolerant or both thermotolerant and osmotolerant and thus were classified as potentially pathogenic strains. To the best of our knowledge, this is the first report on the molecular characterization at the genotype level of Acanthamoeba strains in Venezuela.

High occurrence of Acanthamoeba genotype T4 in soil sources from Bolívar State, Venezuela.

Pathogenic strains of Acanthamoeba are causative agents of keratitis and encephalitis that often may end fatal in humans and other animals. In the present study, twenty-seven soil samples were collected in the Bolivar State in Venezuela and checked for the presence of Acanthamoeba. Samples were cultivated onto 2% non-nutrient agar plates seeded with a layer of heat killed E. coli. Amplification by PCR and sequencing of the DF3 region of the 18S rDNA of Acanthamoeba was carried out in order to confirm morphological identification of the amoebae. Furthermore, Acanthamoeba spp. was isolated from 51.8% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotype T4 in all samples. To the best of our knowledge, this is the first report of genotype T4 in soil sources from Venezuela. Further studies should be carried out in this State and in the country in order to determine the current occurrence of Acanthamoeba in Venezuelan environments.

Vannellid Species Isolated from Freshwater Source in a Park in Jamaica, West Indies.

Free-living amoebae (FLA) occupy a wide range of freshwater, marine, and soil habitats, and are opportunistic pathogens in human beings. While Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are well-known opportunistic organisms, Vannella epipetala is nonpathogenic. Sediments were collected from a freshwater source from a park in Jamaica to investigate the presence of FLA. Acanthamoeba and Naegleria spp. were not recovered; however, a Vannellid species identified by microscopy and PCR analysis as V. epipetala was isolated. These nonpathogens pose a threat to human beings as they may act as Trojan horses for microsporidian parasites and other pathogens, thereby facilitating their transmission to human beings.

Isolation and molecular characterization of Acanthamoeba genotypes in recreational and domestic water sources from Jamaica, West Indies.

Free living amoebae (FLA) are amphizoic protozoa that are ubiquitous in nature. Infection with FLA may result in neurological, ocular and skin infections. Exposure to Acanthamoeba occurs frequently through water contact and knowledge of the presence of the organisms in water sources is important in understanding transmission dynamics. The distribution of Acanthamoeba was studied in recreational and domestic water samples collected from across Jamaica. Morphological assessment and polymerase chain reaction revealed Acanthamoeba spp. isolates in 50.6% (42/83) and 17.3% (14/81) of recreational and domestic water, respectively. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T3, T4, T5, T10 and T11 corresponding to Acanthamoeba spp: A. griffini, A. triangularis, A. lenticulata, A. culbertsoni and A. hatchetti. Moreover, T4 was the most frequently isolated genotype in both recreational and domestic water. Thermotolerance and osmotolerance assays indicated that most isolates were potentially pathogenic. This is the first report of T3 and T10 genotypes in the Caribbean and the first report of these Acanthamoeba spp. in Jamaican waters. The study shows that there is potential risk of infection to contact wearers who practise poor lens care. Further, Acanthamoeba should be considered as a cause of neurological infections in Jamaica.

Isolation and Molecular Characterization of Acanthamoeba Strains from Dental Units in Costa Rica.

Free-living amoebae are protozoa widely distributed in nature, which can be found in a variety of environments. Four genera are recognized as causal agents of infections in humans and animals: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. In this study, the presence of Acanthamoeba in dental units was determined and the isolates obtained were molecularly characterized; osmotolerance and thermotolerance assays were also performed to evaluate multiplication under these conditions, frequently associated with pathogenicity. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 14% of the units sampled. Osmotolerance and thermotolerance tests were positive for more than 80% of the isolates. Up to date, this is the first study that reports the detection, identification, and genotyping of Acanthamoeba isolated from dental units in Costa Rica and even in Latin-America. Further assays to determine the potential pathogenicity of these Acanthamoeba isolates are underway.

Isolation and genotyping of acanthamoeba strains from soil sources from Jamaica, West Indies.

Acanthamoeba spp. are opportunistic pathogens that are ubiquitous in nature. Many species of this genus are responsible for a fatal encephalitis and keratitis in humans and other animals. Seventy-two soil samples were collected from the parishes across Jamaica and assessed for the presence of Acanthamoeba spp. Cultivation was carried out on non-nutrient agar plates seeded with heat killed Escherichia coli. PCR and sequencing of the DF3 region were carried out in order to genotype the isolated strains of Acanthamoeba. Thermotolerance and osmotolerance assays were utilized to investigate the pathogenic potential of the Acanthamoeba isolates. Acanthamoeba spp. was isolated from 63.9% of soil samples. Sequencing of the DF3 region of the 18S rDNA resulted in the identification of genotypes T4, T5, and T11. T4 genotype was most frequently isolated. Most isolates were thermotolerant or both thermotolerant and osmotolerant, indicating that they may present the potential to cause disease in humans and other animals.

Morphological Features and In Vitro Cytopathic Effect of Acanthamoeba griffini Trophozoites Isolated from a Clinical Case.

Light and transmission electron microscopy observations are reported on the structure and in vitro cytopathic effect of Acanthamoeba griffini trophozoites isolated from a clinical case. Live trophozoites were moderately active with a remarkable pleomorphism which changed from ovoid to quite elongated shapes. When moving, amoebae formed cytoplasmic projections such as wide lamellae and acanthopodia of diverse size and thickness which contain a significant amount of actin. Ultrastructurally, the cytoplasm showed the main organelles found in other free-living amoebae. Coincubation of trophozoites with MDCK cell monolayers resulted in a local damage to target cells after 24 h of interaction, suggesting that the cytopathic effect is contact-dependent. By transmission electron microscopy, amoebae appeared to engulf small portions of the MDCK cells; however, the cells that were not in contact with trophozoites had an unaltered morphology. When epithelial monolayers were incubated with conditioned medium for 24 h, small areas of cell injury were also observed. The phylogenetical analysis as well as the sequencing of the acquired amplified product for the DF3 region of the amoebae isolate confirmed that it belongs to genotype T3, which includes other pathogenic amoebae; besides the activity of two drugs currently used against Acanthamoeba was tested on A. griffini.

Isolation and molecular characterization of Acanthamoeba and Balamuthia mandrillaris from combination shower units in Costa Rica.

Free living amoebae (FLA) are ubiquitous protozoa, which may behave as parasites under certain conditions. Four genera are recognized as causal agents of infections in humans and animals: Naegleria, Sappinia, Acanthamoeba and Balamuthia. This work determines the presence of FLA in combination shower units and employs molecular biology for the characterization of isolates. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 30% of the units sampled. In addition to Acanthamoeba cysts, trophozoites with morphological characteristics similar to Balamuthia were identified. PCR assay using the mitochondrial 16S rRNA gene as a target confirmed the identification of the amoeba as Balamuthia mandrillaris. Up to date, this is the first report of the isolation of B. mandrillaris in Central America and the fifth report worldwide.

The isolation of Balamuthia mandrillaris from environmental sources from Peru.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding.

Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima, Peru.

In March 2010, a 35 year-old HIV/AIDS female patient was admitted to hospital to start treatment with Highly Active Antiretroviral Therapy (HAART) since during a routine control a dramatic decrease in the CD4(+) levels was detected. At this stage, a nasal swab from each nostril was collected from the patient to include it in the samples for the case study mentioned above. Moreover, it is important to mention that the patient was diagnosed in 2009 with invasive pneumococcal disease, acute cholecystitis, pancreatitis and pulmonary tuberculosis. The collected nasal swabs from both nostrils were positive for Vermamoeba vermiformis species which was identified using morphological and PCR/DNA sequencing approaches. Basic Local Alignment Search Tool (BLAST) homology and phylogenetic analysis confirmed the amoebic strain to belong to V.vermiformis species. Molecular identification of the Mycobacterium strain was carried out using a bacterial universal primer pair for the 16S rDNA gene at the genus level and the rpoB gene was amplified and sequenced as previously described to identify the Mycobacterium species (Shin et al., 2008; Sheen et al., 2013). Homology and phylogenetic analyses of the rpoB gene confirmed the species as Mycobacterium chelonae. In parallel, collected swabs were tested by PCR and were positive for the presence of V.vermiformis and M.chelonae. This work describes the identification of an emerging bacterial pathogen,M.chelonae from a Free-Living Amoebae (FLA) strain belonging to the species V.vermiformis that colonized the nasal cavities of an HIV/AIDS patient, previously diagnosed with TB. Awareness within clinicians and public health professionals should be raised, as pathogenic agents such as M.chelonae may be using FLA to propagate and survive in the environment.