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.

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.

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.

Acanthamoeba culbertsoni: Electron-Dense Granules in a Highly Virulent Clinical Isolate.

The virulence of various amoebic parasites has been correlated with the presence of electron-dense granules (EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long-term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long-term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni.

Acanthamoeba genotypes T3 and T4 as causative agents of amoebic keratitis in Mexico.

Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.

Contaminación bacteriana en el agua de unidades dentales de una institución de salud pública / Bacterial contamination in dental unit water from a public health institution

Se evaluó la calidad bacteriológica del agua de 8 clínicas de una institución de salud pública (ISSSTE) de 2 zonas de la ciudad de México, determinándose coliformes fecales y coliformes totales, por medio de la técnica de filtro de membrana, realizándose en 2 diferentes periodos y 3 puntos de muestreo (jeringa triple, llena vasos y suministro) en 9 unidades dentales. De las 8 clínicas analizadas 7 presentaron contaminación bacteriana. Perteneciendo 4 a la zona norte y 3 a la zona sur. El problema de la contaminación en la zona norte se debe principalmente a la mala calidad del agua de suministro la cual en esta zona provenía en su mayoría de las botellas del sistema abastecedor de agua. La otra causa de contaminación se debió a la deficiente o nula desinfección de la jeringa triple. En la zona sur 3 de las 4 clínicas en general presentaron baja contaminación bacteriana solo en el primer periodo, lo cual se puede atribuir a la mejor calidad del agua proveniente de los suministros. Se concluye que los factores determinantes de la presencia de contaminación bacteriana en el agua utilizada en las unidades dentales de ambas zonas fueron: la calidad del agua de suministro y la inadecuada desinfección de la jeringa triple. Mantener una buena calidad de agua en las unidades dentales es muy importante, pues el líquido entra directamente en contacto con la mucosa de cavidad oral, estructuras dentarias, sangre y saliva del paciente, evitando procesos infecciosos que podrían poner en riesgo su salud integral

It was assessed the bacteriological quality of water in 8 clinics of a public health institution (ISSSTE) in 2 areas of Mexico City, determined fecal coliform and total coliform, by means of the technique of membrane filter. There were made 2 different periods and 3 sampling points (syringe triple, flood glasses and supply) in 9 dental units. Of the 8 clinics analyzed 7 presented bacterial pollution. 4 Belonged to the north and 3 to the south zone. The problem of pollution in the north zone is mainly due to the poor water quality of supply which in this area came mostly from the bottles of the flush system. The other source of pollution was due to the poor or non-existent disinfection of the triple syringe. In the south zone in general 3 of 4 clinics presented low bacterial pollution only in the first period, which can be attributed to the better quality of water from the supplies. It concludes that the determining factors in the presence of bacterial pollution in water used in dental units of both areas were: the quality of water supply and inadequate disinfection of the triple syringe. Maintain a good water quality in dental units is very important, because liquid makes direct contact with the mucous membrane of oral cavity, dental structures, blood and saliva of the patient, avoiding infectious processes that could put in risk their integral health

Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City.

A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis.

Amibas de vida libre y anemia aplástica grave en niños / Aplastic anemia and free-living amoeba in two children

Introducción. Las amibas de vida libre habitualmente viven como fagótrofas en el agua y suelo donde se alimentan de bacterias; también pueden producir infecciones del sistema nervioso central y otros tejidos en seres humanos y animales. La anemia aplástica es una entidad caracterizada por pancitopenia secundaria a disminución de la producción en médula ósea de todos los elementos formes de la sangre y ausencia de enfermedad primaria que infiltre, reemplace o anule la hematopoyesis activa. Casos clínicos. Se presentan 2 niños con historia de sangrados, pancitopenia y el diagnóstico de anemia aplástica grave por biopsia y aspirado de médula ósea, que finalmente fallecieron. Se muestran los hallazgos de amibas de vida libre en la médula. Conclusión. En estos casos no se encontró agente causal de la anemia aplástica, y se sugiere a las amibas como oportunistas del padecimiento.