Anaerobic Metabolism in T4 Acanthamoeba Genotype.

Members of the genus Acanthamoeba are of the most common protozoa that has been isolated from a variety of environment and affect immunocompromised individuals, causing granulomatous amoebic encephalitis and skin lesions. Acanthamoeba, in immunocompetent patients, may cause a keratitis related to corneal microtrauma. These free-living amoebas easily adapt to the host environment and wield metabolic pathways such as the energetic and respiratory ones in order to maintain viability for long periods. The energetic metabolism of cysts and trophozoites remains mostly unknown. There are a few reports on the energetic metabolism of these organisms as they are mitochondriate eukaryotes and some studies under aerobic conditions showing that Acanthamoeba hydrolyzes glucose into pyruvate via glycolysis. The aim of this study was to detect the energetic metabolic pathways with emphasis on anaerobic metabolism in trophozoites of three isolates of Acanthamoeba sp belonging to the T4 genotype. Two samples were collected in the environment and one was a clinical sample. The evaluation of these microorganisms proceeded as follows: rupture of trophozoites (7.5 × 103 parasites/ml) and biochemical analysis with high performance liquid chromatography and spectrophotometry. The anaerobic glycolysis was identified through the detection of glucose, pyruvate, and lactate. The protein catabolism was identified through the detection of fumarate, urea, and creatinine. The fatty acid oxidation was identified through the detection of acetate, beta-hydroxybutyrate, and propionate. The detected substances are the result of the consumption of energy reserves such as glycogen and lipids. The anaerobic glycolysis and protein catabolism pathways were observed in all three isolates: one clinical and two environmental. This study represents the first report of energetic pathways used by trophozoites from different isolates of the T4 genotype Acanthamoeba.

Experimental infection of T4 Acanthamoeba genotype determines the pathogenic potential.

T4 is the Acanthamoeba genotype most related to cases of granulomatous amoebic encephalitis (GAE) in immunocompromised patients and of keratitis in contact lens wearers. The determination of the pathogenic potential of Acanthamoeba clinical and environmental isolates using experimental models is extremely important to elucidate the capacity of free-living organisms to establish and cause disease in hosts. The aim of this study was to compare and evaluate the histopathology and culture between two different routes of experimental infection of T4 Acanthamoeba isolated from environmental and clinical source in mice (intracranial and intraperitoneal). Swiss isogenic healthy mice were inoculated with 10(4) trophozoites by intracranial (IC) and intraperitoneal (IP) routes and observed during 21 days. The brains from animals inoculated by the IC route were collected and from the animals of the IP inoculation group, the brains, livers, kidneys, spleens, and lungs were removed. The organs were prepared and appropriately divided to be evaluated with histopathology and culture. There was no significant difference between the inoculation routes in terms of isolates recovery (χ(2) = 0.09; p = 0.76). In the IC group, isolate recovery rate was significantly higher in histopathology than the one achieved by culture (χ(2) = 6.45; p < 0.01). Experimental infection revealed that all isolates inoculated could be considered invasive because it was possible to recover evolutive forms of Acanthamoeba in both routes. This work represents the first in vivo pathogenicity assay of primary isolation source in Central region of Brazil showing in vivo pathogenicity and hematogenous spread capacity of these protozoa, improving the knowledge on free-living amoebae isolates.

Occurrence and characterization of Acanthamoeba similar to genotypes T4, T5, and T2/T6 isolated from environmental sources in Brasília, Federal District, Brazil.

Species of Acanthamoeba can cause keratitis and brain infections. The characterization of environmental isolates is necessary to analyze the risk of human infection. We aimed at identifying and genotyping Acanthamoeba isolates from soil, swimming pools, and water features in Brasília, Federal District, Brazil, as well as determining their physiological characteristics and pathogenic potential. Among the 18 isolates studied, eight were similar to genotype T5, five to T4, and one to T2/T6, classified by the sequence analysis of 18S rDNA. Genotypes of four isolates were not determined. Ten isolates (55%) grew at 37 °C and seven (39%) grew in media with 1.5M mannitol, which are the physiological parameters associated with pathogenic Acanthamoeba; also, four isolates from swimming pools presented high pathogenic potential. Our results indicate a widespread distribution of potentially pathogenic Acanthamoeba T4, T5, and T2/T6 in different environmental sources in Brasília, revealing the potential risk of human infection and the need of preventive measures.

The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome.

We report an Acanthamoeba keratitis case associated with the use of contact lens in a 28-year-old female from Brasília, Brazil. Samples from corneal scraping and contact lens case were used for culture establishment, PCR amplification, and partial sequencing (fragments of ~400kb) of small subunit rDNA; both culture and PCR were positive. The sequence analyses of the cornea and of isolates from the contact lens case showed similarity with the T4 genotype. To the best of our knowledge, this is the first report of T4 Acanthamoeba keratitis case from the Midwest region of Brazil.

Occurrence of potentially highly pathogenic free-living amoebae in ready made salads from restaurants in Jatai, Goias, Brazil

Free-living amoebae (FLA) are part of a group of protozoa found worldwide and in the most diverse environments. They resist various temperatures and disinfection methods, and are a risk to human health. Pathogenic strains grow at high temperatures and under hyperosmolarity conditions. Some FLA genera are mainly related to primary amoebic meningoencephalitis (PAM), skin ulcerations, corneal lesions, kidney and lung infections and keratitis. Therefore, studies that assess the pathogenic potential of FLA are public health issues of great concern. We aimed to evaluate the pathogenic potential of FLA isolated in salads from restaurants in vitro, using osmotolerance and thermotolerance tests. Forty-five isolates were used from ready-made salads purchased in restaurants in Jatai, Goias. Twelve isolates subjected to the osmotolerance test (26.6%) showed growth in 0.5 M mannitol, 18 (40.0%) in 1.0 M mannitol and 16 (35.5%) in 1.5 M mannitol, 13 (28.8%) isolates did not show growth. Four isolates that underwent the thermotolerance test (8.9%) showed growth at 25°C, 8 (17.8%) showed growth at 30°C, 3 (6.7%) showed growth at 37°C and 30 (66.7%) did not show growth. With the indices obtained in the present study, we concluded that 15.6% of the isolates were osmotolerant and thermotolerant. Our findings highlight a public health problem once these FLA are associated with harboring or being harbored by microorganisms responsible for diseases such as diarrhea and meningitis. Measures are required to improve food hygiene and so avoid FLA-related health problems.

Occurrence of parasites in salads in restaurants in Aparecida de Goiânia, Goiás, Brazil

Parasitoses are the most widespread diseases in the world. They are transmited via contaminated water or food. Considering that the daily consumption of vegetables is estimated at 142g per person, the purpose of this study was to evaluate the occurrence of parasites in salads available for consumption in restaurants in Aparecida de Goiânia, Goiás State. Salad samples were collected from the restaurants and parasitological analysis was performed using the Willis, Hoffman, Faust and Ziehl Neelsen techniques as well as cultures for the isolation of free-living amoebae. 51 samples were analyzed, 16 (31.4%) were positive. The parasites detected were: Acanthamoeba spp. in 12 (23.5%); free-living larvae, Schistosoma mansoni and Entamoeba coli in 1 (2.0%); Endolimax nana in 2 (3.9%). The PCR technique determined that 17.6% of the samples presented Toxoplasma gondii DNA. These techniques evidenced that the salad samples presented parasite contamination not only in the restaurants with the lowest price per Kg, but also in the most expensive ones. Therefore, in addition to effective sanitary surveillance, prophylactic measures are necessary regarding suppliers, handlers and restaurant owners to prevent the spread of these and other parasites.

The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome

Abstract We report an Acanthamoeba keratitis case associated with the use of contact lens in a 28-year-old female from Brasília, Brazil. Samples from corneal scraping and contact lens case were used for culture establishment, PCR amplification, and partial sequencing (fragments of ~400kb) of small subunit rDNA; both culture and PCR were positive. The sequence analyses of the cornea and of isolates from the contact lens case showed similarity with the T4 genotype. To the best of our knowledge, this is the first report of T4 Acanthamoeba keratitis case from the Midwest region of Brazil.