Agar dehydration: a simple method for long-term storage of Acanthamoeba spp. collection at room temperature.

This study describes dehydration of agar containing cysts as a novel and inexpensive method for long-term storage of Acanthamoeba spp. collections at room temperature. Five hundred microliters of axenically cultured Acanthamoeba spp. trophozoites (106 cells/mL) in PYG media or 150 µl of amoeba suspension (106 cells or cysts/mL) from monoxenic plate culture was spread onto the surface of non-nutritive agar (NNA, 2-3-mm thick) without or with a layer of heat-inactivated Escherichia coli, respectively. The plates were sealed and incubated at 30 °C. After the encystment, the Parafilm® was removed, and the plates were kept at the same temperature until the NNA was completely dehydrated. The dehydrated cyst-containing NNA was cut in rectangles and stored in airtight tubes at room temperature for up to 3 years. Cyst viability was assessed by inoculating them in fresh NNA with a layer of E. coli and in PYG followed by incubation at 30 °C. One hundred percent of samples from all specimens (19) stored over the 3 years allowed new cultures to be re-established; however, two strains showed reduced viability, at 66.7% and 62.5%, after 2 years of room temperature storage. One hundred percent of the cyst samples produced axenically and maintained in dry NNA allowed the re-establishment of axenic cultures through direct incubation in PYG, with excystment occurring within 24 or 48 h. For the first time, we report the dehydration of cyst-containing agar as an economical and effective method for the long-term storage of Acanthamoeba spp. collections at room temperature. It enables the creation of large collections using reduced space and economical transport of Acanthamoeba strains, in addition to allowing better organization of the collection.

Global prevalence of free-living amoebae in solid matrices - A systematic review with meta-analysis.

The ubiquitous free-living amoebae (FLA) are microorganisms of significant medical, sanitary, and ecological importance. However, their characterization within solid matrices such as soil, dust, sediment, mud, sludge, and compost remain to be systematized. In this study, we conducted a systematic review with meta-analysis to explore the global distribution of FLA in solid matrices. From the analysis of 104 out of 4,414 scientific articles retrieved from different databases, it was found that the general global prevalence of FLA in solid matrices was of 55.13% (95% confidence interval (CI) 49.32-60.94). Specifically, FLA prevalence was high in soil (72.40%, 95% CI 69.08-75.73), sediment (57.91%, 95% CI 50.01-65.81), mud (52.90%, 95% CI 24.01-81.78), dust (48.60%, 95% CI 43.00-54.19), and sewage sludge (40.19%, 95% CI 30.68-49.70). In aerosols it was comparatively lower (17.21%, 95% CI 12.76-21.66). Acanthamoeba spp. (52.23%) and Hartmanella/Vermamoeba spp. (36.06%) were found to be more prevalent, whereas Naegleria spp. (34.98%) and Balamuthia spp. (27.32%) were less prevalent. The distribution of the highest global prevalence values for species of Acanthamoeba spp., considering different publication periods of the studies, is as follows: A. hatchetti (51.46%), A. rhysodes (47.49%), A. polyphaga (36.37%), A. culbertsoni (34.31%), A. castellanii (34.21%), and A. lenticulata (32.82%). For other FLA species, the distribution is: Hartmannella/Vermamoeba vermiformis (91.57%), Naegleria fowleri (42.32%), Naegleria gruberi (32.39%), and Balamuthia mandrillaris (25%). The most prevalent Acanthamoeba genotypes were T4 (33.38%) and T3 (23.94%). Overall, the global prevalence of FLA in solid matrices is as high as or greater than that reported in water by previous systematic reviews. Thus, actions aimed at reducing exposure to FLA or exploring their ecological dynamics should consider not only water but also the various solid matrices. The finding outlined here can provide valuable insights for such actions, e.g., informing on the level of exposure to FLA, or on the microbial biodiversity of specific environmental compartments.

Prevalence of free-living amoebae in swimming pools and recreational waters, a systematic review and meta-analysis.

Free-living amoebae (FLA) are cosmopolitan microorganisms known to be pathogenic to humans who often have a history of contact with contaminated water. Swimming pools and recreational waters are among the environments where the greatest human exposure to FLA occurs. This study aimed to determine the prevalence of FLA in swimming pools and recreational waters, through a systematic review and meta-analysis that included studies published between 1977 and 2022. A total of 106 studies were included and an overall prevalence of FLA in swimming pools and recreational waters of 44.34% (95% CI = 38.57-50.18) was found. Considering the studies published up to 2010 (1977-2010), between 2010 and 2015, and those published after 2010 (> 2010-2022), the prevalence was 53.09% (95% CI = 43.33-62.73) and 37.07% (95% CI = 28.87-45.66) and 45.40% (95% CI = 35.48-55.51), respectively. The highest prevalence was found in the American continent (63.99%), in Mexico (98.35%), and in indoor hot swimming pools (52.27%). The prevalence varied with the variation of FLA detection methods, morphology (57.21%), PCR (25.78%), and simultaneously morphology and PCR (43.16%). The global prevalence by genera was Vahlkampfia spp. (54.20%), Acanthamoeba spp. (33.47%), Naegleria spp. (30.95%), Hartmannella spp./Vermamoeba spp. (20.73%), Stenamoeba spp. (12.05%), and Vannella spp. (10.75%). There is considerable risk of FLA infection in swimming pools and recreational waters. Recreational water safety needs to be routinely monitored and, in case of risk, locations need to be identified with warning signs and users need to be educated. Swimming pools and artificial recreational water should be properly disinfected. Photolysis of NaOCl or NaCl in water by UV-C radiation is a promising alternative to disinfect swimming pools and artificial recreational waters.

Clinical and molecular diagnosis of Acanthamoeba keratitis in contact lens wearers in southern Brazil reveals the presence of an endosymbiont.

Acanthamoeba keratitis (AK) is an infection that is mostly observed in contact lens wearers. It is often misdiagnosed causing delays in the administration of the correct treatment. The aim of this study was to report the outcome of clinical and molecular diagnosis of AK cases during the summer of 2019 in the southern region of Brazil. Three suspected cases of AK were discovered after an ophthalmic examination at a public hospital in the city of Porto Alegre. These cases were then confirmed through laboratory diagnosis (cell culture and molecular analysis by PCR and sequencing). In each of the three clinical sample cell cultures of corneal scraping and molecular analysis confirmed the presence of Acanthamoeba spp., all belonging to the morphological group II and to the genotype T4, which is the most common genotype associated with AK. In addition, Acanthamoeba spp. isolated from one of the clinical samples was found to harbor the Candidatus Paracaedibacter acanthamoeba, a bacterial endosymbiont. The presence of Ca. Paracaedibacter acanthamoeba in clinical isolates requires further research to reveal its possible role in the pathogenicity of Acanthamoeba infections.

Naegleria fowleri-associated meningoencephalitis in a cow in Southern Brazil-first molecular detection of N. fowleri in Brazil.

Naegleria fowleri, a free-living and thermophilic ameba, is the etiological agent of primary amebic meningoencephalitis (PAM). PAM is a rare and highly fatal neurologic disease in humans, and has been rarely documented in animal species. This report describes the pathological and etiological findings of a fatal case of N. fowleri-associated meningoencephalitis in a cow in Southern Brazil. Microscopic findings were consistent with severe, multifocal, hemorrhagic, and necrosuppurative meningoencephalitis associated with a large number of amebic trophozoites compatible with N. fowleri. Brain samples subjected to molecular assays generated a 315 bp fragment, which presented 99% identity with a N. fowleri sequence previously deposited in GenBank. This is the first study reporting the molecular detection of N. fowleri in a case of cattle meningoencephalitis in Latin America, and the obtained sequence represents the first GenBank deposit of N. fowleri identified in Brazil to this day. Additionally, the case reported is the second occurrence of N. fowleri-associated disease in the same city, drawing attention to the local importance of infection by this ameba and potential risk for human infections.

Impact of nicotine-induced green tobacco sickness on DNA damage and the relation with symptoms and alterations of redox status in tobacco farmers.

During the harvest period, tobacco workers are exposed to nicotine and it is known that absorption of the alkaloid via the leaves causes green tobacco sickness (GST). We investigated if GST and its symptoms are associated with DNA damage and alterations of the redox status. DNA damage was measured in lymphocytes of tobacco workers and controls (n = 40/group) in single cell gel electrophoresis assays. Exposure to nicotine was determined by plasma cotinine measurements, alterations of the redox status by quantification of the total antioxidant capacity (TEAC) and of thiobarbituric acid reactive substances (TBARS). The symptoms of GTS included nausea, abdominal cramps, headache, vomiting and dizziness, and 50% of the workers had more than one symptom. Cotinine levels were enhanced in the workers (111 ng/mL); furthermore, the extent of DNA damage was ca. 3-fold higher than in the controls. This effect was more pronounced in participants with GST compared to healthy nicotine exposed workers and increased in individuals with specific symptoms (range 22-36%). TBARS levels did not differ between workers and unexposed controls, while TEAC values were even increased (by 14.3%). Contact with nicotine present in tobacco leaves causes GTS and leads to damage of the DNA; this effect is more pronounced in workers with GTS symptoms and is associated with alterations of the redox status. Damage of the genetic material which was found in the workers may lead to adverse long-term effects that are caused by genomic instability such as cancer and accelerated ageing.

Acanthamoeba keratitis in Porto Alegre (southern Brazil): 28 cases and risk factors.

The increasing use of contact lenses worldwide has led to an increase in cases of Acanthamoeba keratitis, which are often associated with inappropriate cleaning of contact lenses and lens cases. This study aimed to retrospectively review 28 cases of Acanthamoeba keratitis in Porto Alegre (southern Brazil) and identify the risk factors and clinical outcomes of affected patients. Most patients had higher education (66.6%), all were users of contact lenses, mostly women (67.9%). Most patients were soft contact lens wearers (66.7%) and 85.7% used multipurpose cleaning solutions. Sixteen patients (64.0%) used to wear contact lenses while swimming and/or bathing. Pain was the most common symptom (92.6%). For treatment, patients used polyhexamethylene biguanide drops (92.6%), propamidine isethionate drops (81.5%), chlorhexidine drops (55.6%), topical corticosteroids (63.0%), and systemic corticosteroids (37.0%). Herpes simplex keratitis was the most common misdiagnosis (72.7%). The majority of patients (76.0%) underwent a corneal transplant to control the disease.