Free-living Amoebae Research Articles

A Narrative Review of Acanthamoeba Isolates in Malaysia: Challenges in Infection Management and Natural Therapeutic Advancements.

Acanthamoeba,a free-living amoeba (FLA) found in diverse ecosystems, poses significant health risks globally, particularly in Malaysia. It causes severe infectious diseases, e.g.,Acanthamoebakeratitis (AK), primarily affecting individuals who wear contact lenses, along with granulomatous amoebic encephalitis (GAE), a rare but often life-threatening condition among immunocompromised individuals. AK has become increasingly prevalent in Malaysia and is linked to widespread environmental contamination and improper contact lens hygiene. Recent studies highlightAcanthamoeba's capacity to serve as a "Trojan horse" for amoeba-resistant bacteria (ARBs), contributing to hospital-associated infections (HAIs). These symbiotic relationships and the resilience ofAcanthamoebacysts make treatment challenging. Current diagnostic methods in Malaysia rely on microscopy and culture, though molecular procedures like polymerase chain reaction (PCR) are employed for more precise detection. Treatment options remain limited due to the amoeba's cyst resistance to conventional therapies. However, recent advancements in natural therapeutics, including using plant extracts such as betulinic acid fromPericampylus glaucusand chlorogenic acid fromLonicera japonica, have shown promising in vitro results. Additionally, nanotechnology applications, mainly using gold and silver nanoparticles to enhance drug efficacy, are emerging as potential solutions. Further, in vivo studies and clinical trials must validate these findings. This review highlights the requirement for continuous research, public health strategies, and interdisciplinary collaboration to address the growing threat ofAcanthamoebainfections in Malaysia while exploring the country's rich biodiversity for innovative therapeutic solutions.

Systemic Acanthamoeba T17 infection in a free-ranging two-toed sloth: case report and literature review of infections by free-living amebas in mammals.

A free-ranging, adult female two-toed sloth (Choloepus hoffmanni) was brought to a wildlife rescue center in Costa Rica with ocular and auricular myiasis and numerous skin lesions. After one month of unsuccessful systemic and topical antimicrobial treatment, the patient died. A postmortem examination was performed, and tissues were examined histologically, confirming disseminated amebic infection with intralesional trophozoites and cysts in the lungs, liver, eye, heart, spleen, and stomach. Immunohistochemistry identified the ameba as Acanthamoeba sp. A multiplex real-time PCR assay, 18S ribosomal DNA PCR, and sequencing performed on formalin-fixed, paraffin-embedded lung tissue confirmed the Acanthamoeba T17 genotype. The Acanthamoeba genus is in the group of free-living amebas that cause infection in humans and animals, and it is ubiquitous in the environment. Acanthamoeba T17 has been isolated from water and soil, but to our knowledge, this genotype has not been implicated in infections of animals previously and has not been reported from Costa Rica. Systemic Acanthamoeba infection has not been described in sloths previously. We provide a comprehensive literature review describing infections by free-living amebas of the genus Acanthamoeba spp., Balamuthia spp., and Naegleria spp. in domestic, zoo, and wild mammals.

Identification of new targets for the diagnosis of cysts (four) and trophozoites (one) of the eye pathogen Acanthamoeba.

Free-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of cysts by microscopy of the eye or of corneal scrapings, using calcofluor-white that unfortunately cross-reacts with fungi and plants. Alternatively, Acanthamoeba infections are diagnosed by identification of trophozoites in cultures of scrapings. Here we showed that rabbit antibodies to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently detect calcofluor-white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rabbit antibodies to the mannose-binding domain of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identifies trophozoites of all 11 ATCC isolates. In summary, four wall proteins and the ManBD appear to be excellent targets for diagnosis of Acanthamoeba cysts and trophozoites, respectively.

The presence of yeasts and bacteria in free-living amoebae isolated from COVID-19 patients: concern for secondary infections

ABSTRACT This study aimed to investigate the presence of SARS-CoV-2, yeasts, and bacteria in isolated free-living amoeba (FLA) from COVID-19 patients. Nasopharyngeal swabs (n = 60) were obtained from COVID-19 patients. After cultivation, morphological characterization, and RNA/DNA extraction, the presence of selected microorganisms was investigated. From 60 COVID-19 samples, 18 (30%) were positive for FLA. Acanthamoeba sp. Naegleria australiensis, Tetramitus sp. and Vermamoeba vermiformis were characterized in 12 (80%), 1 (6.66%), 2 (13.33%), and 7 (38.88%) of samples, respectively. SARS-CoV-2 RNA was not detected in FLA. Candida albicans, C. tropicalis, and C. parapsilosis were detected in (11/18; 61.11%), (3/18; 16.67%), and (3/18; 16.67%) of samples, respectively. Geotrichum candidum was detected in 10/18 (55.55%) of samples. Streptococcus spp. and Staphylococcus spp. were identified in 16/18 (88.88%) and 3/18 (16.67%), respectively. The presence of yeasts and bacteria signifies the possible role of FLA in distribution of secondary infections in susceptible patients.

First report on the long-term viability of Acanthamoeba species in unpreserved environmental freshwater samples stored at room temperature

ABSTRACT Acanthamoeba is an opportunistic, free-living amoeba ubiquitous in the environment. Despite reports of its wide distribution in the Philippines’ freshwater resources, more information on the long-term viability of the Acanthamoeba species is needed. This study aimed to define the long-term viability of Acanthamoeba species in unpreserved environmental freshwater samples after 3 years of storage at room temperature. Stored water samples from 15 study sites were filtered through a 1.2-μm pore size glass microfiber filter, cultured in non-nutrient agar (NNA) lawned with Escherichia coli, and observed for amoebic growth for 14 days using light microscopy. Isolates from positive NNA culture were subjected to polymerase chain reaction (PCR) using JDP1 and JDP2 Acanthamoeba-specific primers. The study site positivity was 33% (5/15). Acanthamoeba genotype T4 and Acanthamoeba lenticulata were isolated from Luzon; Acanthamoeba divionensis was isolated from Visayas; and Acanthamoeba sp. and genotype T20 were isolated from Mindanao. The long-term viability of Acanthamoeba species is an added risk factor for the sustained contamination of aquatic resources and other sample matrices. This heightens the risk of transmission to humans and animals. This study demonstrated that water samples fated for Acanthamoeba studies can be stored unpreserved at room temperature for several years.

Potentially Pathogenic Free-Living Amoebae Isolated from Soil Samples from Warsaw Parks and Squares.

Free-living amoebae (FLA) are prevalent in diverse environments, representing various genera and species with different pathogenicity. FLA-induced infections, such as the highly fatal amoebic encephalitis, with a mortality rate of 99%, primarily affect immunocompromised individuals while others such as Acanthamoeba keratitis (AK) and cutaneous amebiasis may affect immunocompetent individuals. Despite the prevalence of FLA, there is a lack of standardized guidelines for their detection near human habitats. To date, no studies on the isolation and identification of FLA in environmental soil samples in Warsaw have been published. The aim of this study was to determine the presence of amoebae in soil samples collected from Warsaw parks and squares frequented by humans. The isolated protozoa were genotyped. Additionally, their pathogenic potential was determined through thermophilicity tests. A total of 23 soil samples were seeded on non-nutrient agar plates (NNA) at 26 °C and monitored daily for FLA presence. From the total of 23 samples, 18 were positive for FLA growth in NNA and PCR (78.2%). Acanthamoeba spp. was the most frequently isolated genus, with a total of 13 positive samples (13/18; 72.2%), and the T4 genotype being the most common. Moreover, Platyamoeba placida (3/18; 16.7%), Stenamoeba berchidia (1/18; 5.6%) and Allovahlkampfia sp. (1/18; 5.6%), also potentially pathogenic amoebae, were isolated. To our knowledge, this is the first report of FLA presence and characterization in the Warsaw area.

Acanthamoeba castellanii trophozoites need oxygen for normal functioning and lipids are their preferred substrate, offering new possibilities for treatment

Acanthamoebae, pathogenic free-living amoebae, can cause Granulomatous Amoebic Encephalitis (GAE) and keratitis, and for both types of infection, no adequate treatment options are available. As the metabolism of pathogens is an attractive treatment target, we set out to examine the energy metabolism of Acanthamoeba castellanii and studied the aerobic and anaerobic capacities of the trophozoites. Under anaerobic conditions, or in the presence of inhibitors of the electron-transport chain, A. castellanii trophozoites became rounded, moved sluggishly and stopped multiplying. This demonstrates that oxygen and the respiratory chain are essential for movement and replication. Furthermore, the simultaneous activities of both terminal oxidases, cytochrome c oxidase and the plant-like alternative oxidase, are essential for normal functioning and replication. The inhibition of normal function caused by the inactivity of the respiratory chain was reversible. Once respiration was made possible again, the rounded, rather inactive amoebae formed acanthopodia within 4 h and resumed moving, feeding and multiplying. Experiments with radiolabelled nutrients revealed a preference for lipids over glucose and amino acids as food. Subsequent experiments showed that adding lipids to a standard culture medium of trophozoites strongly increased the growth rate. Acanthamoeba castellanii trophozoites have a strictly aerobic energy metabolism and β-oxidation of fatty acids, the Krebs cycle, and an aerobic electron-transport chain coupled to the ATP synthase, producing most of the used ATP. The preference for lipids can be exploited, as we show that three known inhibitors of lipid oxidation strongly inhibited the growth of A. castellanii. In particular, thioridazine and perhexiline showed potent effects in low micromolar concentrations. Therefore, this study revealed a new drug target with possibly new options to treat Acanthamoeba infections.

The potential of nanocomposites (patuletin-conjugated with gallic acid-coated zinc oxide) against free-living amoebae pathogens.

Free-living amoebae infections are on the rise while the prognosis remains poor. Current therapies are ineffective, and there is a need for novel effective drugs which can target Naegleria, Balamuthia, and Acanthamoeba species. In this study, we determined the effects of a nano-formulation based on flavonoid patuletin-loaded gallic acid functionalized zinc oxide nanoparticles (PA-GA-ZnO) against Acanthamoeba, Balamuthia, and Naegleria trophozoites. Characterization of the nano-formulation was accomplished utilizing analytical tools, namely Fourier-transform infrared spectroscopy, drug entrapment efficiency, polydispersity index, dimensions, and surface morphologies. Anti-amoebic effects were investigated using amoebicidal assay, cytopathogenicity assay, and cytotoxicity of the nano-formulation on human cells. The findings revealed that nano-formulation (PA-GA-ZnO) displayed significant anti-amoebic properties and augmented effects of patuletin alone against all three brain-eating amoebae. When tested alone, patuletin nano-formulations showed minimal toxicity effects against human cells. In summary, the nano-formulations evaluated herein depicts efficacyversusAcanthamoeba, Balamuthia, and Naegleria. Nonetheless, future studies are needed to comprehend the molecular mechanisms of patuletin nano-formulations versus free-living amoebae pathogens, in addition to animal studies to determine their potential value for clinical applications.

A Comprehensive Review on Acanthamoeba Keratitis: An Overview of Epidemiology, Risk Factors, and Therapeutic Strategies.

Acanthamoeba keratitis (AK) is a rare but severe corneal infection caused by the free-living amoeba, Acanthamoeba, which is ubiquitously present in the environment. This condition predominantly affects contact lens wearers but can also occur in non-lens users, particularly those exposed to contaminated water or with compromised immune systems. AK is characterized by progressive corneal inflammation, epithelial defects, and ulceration, which can lead to significant visual impairment or blindness if not promptly diagnosed and treated. This review aims to provide a comprehensive overview of AK by synthesizing current knowledge on its epidemiology, risk factors, pathophysiology, clinical manifestations, diagnostic approaches, and therapeutic strategies. The review also highlights preventive measures and public health strategies to reduce the incidence of this debilitating condition. A detailed examination of existing literature was conducted, focusing on the global incidence of AK, demographic trends, and various risk factors such as contact lens use, environmental exposures, and immunity status. The review also delves into the pathophysiology of Acanthamoeba infection, the host immune response, and the challenges in distinguishing AK from other forms of infectious keratitis. Therapeutic strategies, including medical and surgical interventions, are analyzed, along with emerging treatments. The global incidence of AK has increased, particularly among contact lens users, due to poor hygiene practices and environmental exposures. Early diagnosis remains challenging, often leading to delayed treatment and poorer outcomes. Biguanides and diamidines are the mainstays of medical therapy, with surgical options considered in advanced cases. Emerging therapies, such as photodynamic therapy and antimicrobial peptides, show promise in enhancing treatment outcomes.AK poses a significant threat to ocular health due to its potential for severe visual impairment and the complexities associated with its diagnosis and treatment. Early recognition, appropriate management, and public health initiatives focused on prevention are crucial for improving patient outcomes. Ongoing research and a collaborative approach among healthcare providers are essential to advancing the understanding and management of AK.

Approaches for Targeting Naegleria fowleri Using Nanoparticles and Artificial Peptides.

Naegleria fowleri is a free-living amoeba which causes primary amoebic meningoencephalitis (PAM). Although PAM is rare, the fatality rate is staggering at over 97%. So, the importance of finding an effective treatment and cure for PAM caused by N. fowleri is a crucial area of research. Existing research on developing novel therapeutic strategies to counter N. fowleri infection is limited. Since the blood-brain barrier (BBB) presents an obstacle to delivering drugs to the site of infection, it is important to employ strategies that can effectively direct the therapeutics to the brain. In this regard, our review focuses on understanding the physiology and mechanisms by which molecules pass through the BBB, the current treatment options available for PAM, and the recent research conducted in the decade of 2012 to 2022 on the use of nanomaterials to enhance drug delivery. In addition, we compile research findings from other central nervous system (CNS) diseases that use shuttle peptides which allow for transport of molecules through the BBB. The approach of utilizing BBB shuttles to administer drugs through the BBB may open up new areas of drug discovery research in the field of N. fowleri infection.

Thermophilic free-living amoebae from water sources of Costa Rica and identification of physicochemical parameters related to the presence of Naegleria

Thermophilic free-living amoebae from water sources of Costa Rica and identification of physicochemical parameters related to the presence of Naegleria

Isolates of Acanthamoeba species in the marine environment in the Philippines.

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acanthamoeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs.

Synthesis and Biological Evaluation of Cyanoacrylamides and 5-Iminopyrrol-2-Ones Against Naegleria fowleri.

Primary amoebic meningoencephalitis is caused by the free-living amoeba Naegleria fowleri. The lack of standardized treatment has significantly contributed to the high fatality rates observed in reported cases. Therefore, this study aims to explore the anti-Naegleria activity of eight synthesized cyanoacrylamides and 5-iminopyrrol-2-ones. Notably, QOET-109, QOET-111, QOET-112, and QOET-114 exhibited a higher selectivity index against Naegleria compared to those of the rest of the compounds. Subsequently, these chemicals were assessed against the resistant stage of N. fowleri, demonstrating activity similar to that observed in the vegetative stage. Moreover, characteristic events of programmed cell death were evidenced, including chromatin condensation, increased plasma membrane permeability, mitochondrial damage, and heightened oxidative stress, among others. Finally, this research demonstrated the in vitro activity of the cyanoacrylamide and 5-iminopyrrol-2-one molecules, as well as the induction of metabolic event characteristics of regulated cell death in Naegleria fowleri.

Diversity of Free-Living Amoebae in New Zealand Groundwater and Their Ability to Feed on Legionella pneumophila.

Free-living amoebae (FLA) are common in both natural and engineered freshwater ecosystems. They play important roles in biofilm control and contaminant removal through the predation of bacteria and other taxa. Bacterial predation by FLA is also thought to contribute to pathogen dispersal and infectious disease transmission in freshwater environments via the egestion of viable bacteria. Despite their importance in shaping freshwater microbial communities, the diversity and function of FLA in many freshwater ecosystems are poorly understood. In this study, we isolated and characterized FLA from two groundwater sites in Canterbury, New Zealand using microbiological, microscopic, and molecular techniques. Different methods for groundwater FLA isolation and enrichment were trialed and optimized. The ability of these isolated FLA to predate on human pathogen Legionella pneumophila was assessed. FLA were identified by 18S metagenomic amplicon sequencing. Our study showed that Acanthamoeba spp. (including A. polyphaga) and Vermamoeba veriformis were the main FLA species present in both groundwater sites examined. While most of the isolated FLA co-existed with L. pneumophila, the FLA populations in the L. pneumophila co-culture experiments predominantly consisted of A. polyphaga, Acanthamoeba spp., Naegleria spp., V. vermiformis, Paravahlkampfia spp., and Echinamoeba spp. These observations suggest that FLA may have the potential to act as reservoirs for L. pneumophila in Canterbury, New Zealand groundwater systems and could be introduced into the local drinking water infrastructure, where they may promote the survival, multiplication, and dissemination of Legionella. This research addresses an important gap in our understanding of FLA-mediated pathogen dispersal in freshwater ecosystems.

Multiplex Real-Time Polymerase Chain Reaction Assay To Detect Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris in Different Water Sources.

Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

Acanthamoebae as a protective reservoir for Pseudomonas aeruginosa in a clinical environment

Pseudomonas aeruginosa is a growing concern in healthcare-associated infections and poses significant risk to those with serious underlying health conditions. The antimicrobial resistance traits of the pathogen and ability to form biofilms make effective mitigation and disinfection strategies difficult. Added to this challenge is the role that free-living amoebae such as Acanthamoeba play in the detection, disinfection and transmission of P.aeruginosa. P. aeruginosa can survive intracellularly within amoebae, which has the potential to limit detectability and permit transmission into high-risk areas. We screened for the presence of Acanthamoeba spp. and P.aeruginosa within a functioning general hospital in Scotland using a culture and molecular approach, noting their presence at several sites over a four-month period, particularly within floor drains connecting patient rooms. In addition, microbiome analysis revealed that amoebae harbour a unique microbial community comprised primarily of Pseudomonas spp. that were not readily detected using microbiome sequencing techniques on environmental swabs. Having demonstrated that both organisms were consistently present in hospital settings, we investigated the relationship between acanthamoeba and P.aeruginosa in the laboratory, showing that (i) acanthamoeba growth rate is increased in the presence of pseudomonas biofilms and viable pseudomonas persist within the amoebae and (ii) hydrogen peroxide-based disinfectants are significantly less effective against an isolate of P.aeruginosa in the presence of acanthamoeba than when the bacteria are incubated alone. These findings suggest that amoebae, and other protists, can influence the detection and persistence of P.aeruginosa in high-risk areas and should be considered when implementing mitigation strategies.

Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amoebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate these agents are potent inhibitors of N. fowleri ENO (NfENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC50 = 0.14 ± 0.04 μM) that was toxic to trophozoites (EC50 = 0.21 ± 0.02 μM) while the reported CC50 was >300 μM. Molecular docking simulation revealed that HEX binds strongly to the active site of NfENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of NfENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the end of the one-week observation, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. However, intranasal HEX delivery was not curative as brains of six of the eight survivors were positive for amoebae. These findings suggest that HEX requires further evaluation to develop as a lead for treatment of PAM.

First Report of Acanthamoeba Genotype T4 from the Newly Formed Tajogaite Volcano Tephra (La Palma, Canary Islands).

The Tajogaite Volcano erupted on the western slope of the Cumbre Vieja mountain range on La Palma Island in the Canary Islands, Spain, in 2021. As one of the multiple consequences of this eruption, a layer of tephra was deposited, to a variable extent, over a large part of the island. Tephra deposits affect all aspects of vegetation recovery, the water cycle, and the long-term availability of volcanic nutrients. Protozoa, including free-living amoeba (FLA), are known to be among the first microorganisms capable of colonizing harsh environments. In the present study, the presence of FLA has been evaluated in the Tajogaite Volcano deposits. Samples of the tephra were collected and incubated at 26 °C on 2% non-nutrient agar plates with a layer of heat-killed E. coli. Morphological features, as well as the DF3 region sequence of the 18S rDNA, confirmed the presence of a T4 genotype strain of Acanthamoeba. Thermotolerance and osmotolerance assays were used to evaluate the strain's pathogenic potential. This strain was considered thermotolerant but poorly osmotolerant. To the best of our knowledge, this is the first report of Acanthamoeba being isolated from a recently erupted volcano.

Brainstorm: A case of granulomatous encephalitis

Background: Free-living amoebas (FLAs) can cause severe and fatal central nervous system infections that are difficult to diagnose. Methods: We present the case of a 74-year-old immunocompetent woman admitted for focal neurological symptoms with enhancing lesions in the right cerebellar hemisphere. A first cerebral biopsy showed granulomatous inflammation, but no microorganisms were identified. After transient clinical improvement, she eventually deteriorated 4 months after initial presentation, with an MRI confirming multiple new masses affecting all cerebral lobes. Results: A second brain biopsy revealed granulomatous and acute inflammation with organisms containing a large central nucleus with prominent karyosome, consistent with FLAs. Immunohistochemical and polymerase chain reaction assays performed at CDC were positive for Acanthamoeba spp, confirming the diagnosis of granulomatous amoebic encephalitis (GAE) caused by Acanthamoeba spp. The patient was treated with combination therapy recommended by CDC, but died a few days later. Upon histopathological rereview, amoebic cysts and trophozoites were identified by histochemical and immunohistochemical methods in the first cerebral biopsy. Conclusion: FLA infections can be challenging to diagnose because of the low incidence, non-specific clinical and radiological presentation, lack of accessible diagnostic tools, and clinicians’ unfamiliarity. This case highlights the importance of recognizing FLA as a potential cause of granulomatous encephalitis, even in the absence of risk factors, as early treatment might be associated with favourable outcomes in case reports. When suspected, CDC laboratories offer tests to confirm the diagnosis promptly.

Surviving chlorinated waters: bleaching sensitivity and persistence of free-living amoebae.

Recent advancements in membrane technologies and disinfection methods have enhanced drinking water quality significantly. However, microorganisms, including free-living amoebae (FLA), persist and pose potential threats to humans. FLA are linked to severe neuro-ophthalmic infections and serve as hosts of pathogenic bacteria. This study examined FLA presence in chlorinated and ultrafiltration drinking water and evaluated chlorine's disinfectant. Of 115 water samples, 21 tested positive for Acanthamoeba sp., Allovahlkampfia sp., and Vermamoeba vermiformis, originating from chlorinated sources. FLA trophozoites withstand temperatures up to 37°C, while the cysts tolerate heat shocks of 60-70°C. Trophozoites are susceptible to 5mg L-1 chlorine, but cysts remain viable at concentrations up to 10mg L-1. FLAs' survival in chlorinated waters is attributed to high cyst tolerance and lower residual chlorine concentrations. These findings highlight the need for ultrafiltration or enhanced chlorination protocols to ensure safer drinking water.