OBJECTIVE: To report real-world data (RWD) on the use of in vivo confocal microscopy (IVCM) in handling cases of suspected Acanthamoeba keratitis (AK) cases at a regional referral center during a 12-year period.
METHODS: Retrospective study of patients with suspected AK presenting at a regional referral center for IVCM in Sweden from 2010 to 2022. Demographics, symptoms, outcomes, and clinical management were analyzed, and IVCM images were interpreted.
RESULTS: Of 74 included patients with suspected AK, 18 (24%) were IVCM-positive, 33 (44%) were IVCM-negative, 15 had inconclusive IVCM results (20.2%), and 8 (11%) were referred for a second opinion based on IVCM, 4 of which were IVCM-positive (5.5%), yielding an overall IVCM-positive rate of 29.5%. Cultures were taken in 38 cases (51%) with only 2 cases (2.7%) culture-positive for AK. Of IVCM-negative cases, cultures were taken in 22 (67%) of cases and 100% of these were AK-negative. IVCM-positive cases had more clinic visits (median 30, P = 0.018) and longer follow-up time (median 890 days, P = 0.009) than IVCM-negative patients, while visual acuity improvement did not differ (P > 0.05). Of IVCM-positive cases, 10 (56%) underwent surgery despite prior anti-amoebic treatment, and 14 (78%) had 3 or more IVCM examinations during follow-up, with cysts (100%), dendritic cells (89%) and inflammatory infiltrate (67%) as the most prevalent features. Longitudinal IVCM indicated improvement in cysts, dendritic cells and subbasal nerves with treatment, while clinical resolution was not always consistent with complete absence of cysts.
CONCLUSIONS: In a real-world setting, IVCM has a high reliability in classifying AK-negative cases, while IVCM detects AK-positive cases more frequently than the gold-standard culture method, leading to its preferential use over the culture method where time or resources are limited. Despite this, a subset of cases are IVCM-inconclusive, the clinical course of referred patients is long requiring many hospital visits, and visual acuity in most cases does not improve with medical treatment alone. Information sharing across centers and standardization of referral and diagnostic routines is needed to exploit the full potential of IVCM in AK patient management.

Acanthamoeba, are ubiquitous eukaryotic microorganisms, that play a pivotal role in recognizing and engulfing various microbes during predation, offering insights into microbial dynamics and immune responses. An intriguing observation lies in the apparent preference of Acanthamoeba for Gram-negative over Gram-positive bacteria, suggesting potential differences in the recognition and response mechanisms to bacterial prey. Here, we comprehensively review pattern recognition receptors (PRRs) and microbe associated molecular patterns (MAMPs) that influence Acanthamoeba interactions with bacteria. We analyze the molecular mechanisms underlying these interactions, and the key finding of this review is that Acanthamoeba exhibits an affinity for bacterial cell surface appendages that are decorated with carbohydrates. Notably, this parallels warm-blooded immune cells, underscoring a conserved evolutionary strategy in microbial recognition. This review aims to serve as a foundation for exploring PRRs and MAMPs. These insights enhance our understanding of ecological and evolutionary dynamics in microbial interactions and shed light on fundamental principles governing immune responses. Leveraging Acanthamoeba as a model organism, provides a bridge between ecological interactions and immunology, offering valuable perspectives for future research.

Chlorocresol has antibacterial and antifungal properties, yet its effectiveness in eradicating Acanthamoeba spp. remains unexplored. Acanthamoeba species trophozoites are usually sensitive to biocides, whereas cysts tend to be more resistant. This study aimed to evaluate the cysticidal activity of chlorocresol against Acanthamoeba polyphaga. Chlorocresol concentrations of 0.02, 0.04, and 0.08% were prepared and A. polyphaga cysts were incubated at room temperature (28-37 C) for 1, 24, 48, and 72 hr at each concentration. Cyst viability was evaluated using trypan blue staining and the percentage of nonviable cysts was calculated. For qualification assays, treated cysts were cultured on nonnutrient agar medium coated with Escherichia coli, incubated at 30 C, observed under a stereomicroscope for 30 days, and inoculated into peptone-yeast extract-glucose medium at 30 C for 72 hr. The results revealed that the A. polyphaga cysts were susceptible to 0.02, 0.04, and 0.08% chlorocresol. Chlorocresol made a significant difference in viability (P < 0.001) compared with the nontreated control for the same incubation time. This is the first study to examine the efficacy of chlorocresol against A. polyphaga cysts and it was highly effective. Chlorocresol could thus serve as an alternative chemical disinfectant for the eradication of A. polyphaga cysts as well as a prophylactic against transmission of other pathogenic microorganisms for which Acanthamoeba species can act as a carrier.

In the context of the virosphere, viral particles can compete for host cells. In this scenario, some viruses block the entry of exogenous virions upon infecting a cell, a phenomenon known as superinfection inhibition. The molecular mechanisms associated with superinfection inhibition vary depending on the viral species and the host, but generally, blocking superinfection ensures the genetic supremacy of the virus\'s progeny that first infects the cell. Giant amoeba-infecting viruses have attracted the scientific community\'s attention due to the complexity of their particles and genomes. However, there are no studies on the occurrence of superinfection and its inhibition induced by giant viruses. This study shows that mimivirus, moumouvirus, and megavirus, exhibit different strategies related to the infection of Acanthamoeba. For the first time, we have reported that mimivirus and moumouvirus induce superinfection inhibition in amoebas. Interestingly, megaviruses do not exhibit this ability, allowing continuous entry of exogenous virions into infected amoebas. Our investigation into the mechanisms behind superinfection blockage reveals that mimivirus and moumouvirus inhibit amoebic phagocytosis, leading to significant changes in the morphology and activity of the host cells. In contrast, megavirus-infected amoebas continue incorporating newly formed virions, negatively affecting the available viral progeny. This effect, however, is reversible with chemical inhibition of phagocytosis. This work contributes to the understanding of superinfection and its inhibition in mimivirus, moumouvirus, and megavirus, demonstrating that despite their evolutionary relatedness, these viruses exhibit profound differences in their interactions with their hosts.IMPORTANCESome viruses block the entry of new virions upon infecting a cell, a phenomenon known as superinfection inhibition. Superinfection inhibition in giant viruses has yet to be studied. This study reveals that even closely related viruses, such as mimivirus, moumouvirus, and megavirus, have different infection strategies for Acanthamoeba. For the first time, we have reported that mimivirus and moumouvirus induce superinfection inhibition in amoebas. In contrast, megaviruses do not exhibit this ability, allowing continuous entry of exogenous virions into infected amoebas. Our investigation shows that mimivirus and moumouvirus inhibit amoebic phagocytosis, causing significant changes in host cell morphology and activity. Megavirus-infected amoebas, however, continue incorporating newly formed viruses, affecting viral progeny. This research enhances our understanding of superinfection inhibition in these viruses, highlighting their differences in host interactions.

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. Herein, we have screened for the presence of Acanthamoeba species and P. aeruginosa within a functioning general hospital in Scotland using a culture and molecular approach, noting their presence at several sites over a 4-month period, particularly within floor drains connecting patient rooms. In addition, microbiome analysis revealed amoebae harbour a unique microbial community comprised primarily of Pseudomonas species 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.

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.

Acanthamoeba keratitis (AK) is a sight-threatening and difficult-to-treat ocular infection. The significant side effects of current AK treatments highlight the urgent need to develop a safe and effective AK medication. In this study, the amoebicidal activity of Iris setosa Pall. ex Link extract (ISE) against Acanthamoeba was examined and its specific amoebicidal mechanism was explored. ISE induced significant morphological changes in Acanthamoeba trophozoites and exhibited amoebicidal activity against A. castellanii and A. polyphaga. ISE was further fractionated into five subfractions by sequential extraction with n-hexane, chloroform, ethyl acetate, n-butanol, and water, and their amoebicidal activities and underlying amoebicidal mechanisms were investigated. The n-butanol subfraction of ISE (ISE-BuOH) displayed selective amoebicidal activity against the Acanthamoeba species with minimal cytotoxicity in human corneal cells (HCE-2). ISE-BuOH triggered apoptosis-like programmed cell death (PCD) in amoebae, characterized by DNA fragmentation, increased ROS production, and caspase-3 activity elevation. ISE-BuOH also demonstrated a partial cysticidal effect against the amoeba species. ISE-BuOH could be a promising candidate in the development of therapeutic drugs for AK.

BACKGROUND: This study aimed at measuring the effect of contact lens hygiene risk factors on the prevalence of contact lens-related keratitis and identifying the specific risk factors to both microbial and non-microbial keratitis independently.
METHODS: A cross-sectional study was conducted at Alexandria Ophthalmology Hospital from May to October 2023. All contact lens wearers attending the outpatient clinic had undergone face-to-face interviews using a standardized validated questionnaire which included demographic data and contact lenses (CLs) hygiene risk factors. Participants were classified into two groups; normal group and keratitis group. Keratitis group was further subdivided into non-microbial and microbial group.
RESULTS: The study included 245 contact lens wearers; 149 normal cases, 50 (20.4%) contact lens-related non-microbial keratitis (CLNK) cases, and 46 (18.8%) contact lens-related microbial keratitis (CLMK) cases. Sharing contact lenses and eye trauma were significant risk factors for both CLNK (p=0.036), (p=0.001) and CLMK (p=0.003), (p=0.017). CLs wear duration for more than 12 hours was associated with an increased risk of CLNK by about 4 times (p=0.030) and overnight wear of contact lenses increased the risk of CLNK by 2.6 times (p=0.030). Showering or swimming in lenses was identified as a significant risk factor for CLMK (p=0.012), moreover washing lenses with tap water increased the risk of CLMK (p=0.030).
CONCLUSIONS: Poor compliance with contact lenses hygiene rules results in a high prevalence of contact lens-related keratitis. Eye trauma and sharing contact lenses were significant hygiene risk factors for both contact lens-related non-microbial keratitis and contact lens-related microbial keratitis.

The cyst wall of the eye pathogen Acanthamoeba castellanii contains cellulose and has ectocyst and endocyst layers connected by conical ostioles. Cyst walls contain families of lectins that localize to the ectocyst layer (Jonah) or the endocyst layer and ostioles (Luke and Leo). How lectins and an abundant laccase bind cellulose and why proteins go to locations in the wall are not known and are the focus of the studies here. Structural predictions identified β-jelly-roll folds (BJRFs) of Luke and sets of four disulfide knots (4DKs) of Leo, each of which contains linear arrays of aromatic amino acids, also present in carbohydrate-binding modules of bacterial and plant endocellulases. Ala mutations showed that these aromatics are necessary for cellulose binding and proper localization of Luke and Leo in the Acanthamoeba cyst wall. BJRFs of Luke, 4DKs of Leo, a single β-helical fold (BHF) of Jonah, and a copper oxidase domain of the laccase each bind to glycopolymers in both layers of deproteinated cyst walls. Promoter swaps showed that ectocyst localization does not just correlate with but is caused by early encystation-specific expression, while localization in the endocyst layer and ostioles is caused by later expression. Evolutionary studies showed distinct modes of assembly of duplicated domains in Luke, Leo, and Jonah lectins and suggested Jonah BHFs originated from bacteria, Luke BJRFs share common ancestry with slime molds, while 4DKs of Leo are unique to Acanthamoeba.IMPORTANCEAcanthamoebae is the only human parasite with cellulose in its cyst wall and conical ostioles that connect its inner and outer layers. Cyst walls are important virulence factors because they make Acanthamoebae resistant to surface disinfectants, hand sanitizers, contact lens sterilizers, and antibiotics applied to the eye. The goal here was to understand better how proteins are targeted to specific locations in the cyst wall. To this end, we identified three new proteins in the outer layer of the cyst wall, which may be targets for diagnostic antibodies in corneal scrapings. We used structural predictions and mutated proteins to show linear arrays of aromatic amino acids of two unrelated wall proteins are necessary for binding cellulose and proper wall localization. We showed early expression during encystation causes proteins to localize to the outer layer, while later expression causes proteins to localize to the inner layer and the ostioles.

OBJECTIVE: To provide an overview on the incidence of Acanthamoeba Keratitis (AK).
CONCLUSIONS: Although being a major and sight-threatening cause of infectious keratitis in the population, a comprehensive assessment of the incidence of this condition is lacking.
METHODS: Incidence of AK was computed as the number of AK eyes, per healthcare center, per year (annualized-center-incidence, or ACI). Two meta-analytical ratios were also calculated: a) the ratio of AK eyes to the count of non-viral microbial keratitis (MK) eyes; b) the ratio of AK eyes to the overall population (i.e., the total number of subjects of a nation or region, as indicated by the authors in each study). Center was defined as the healthcare facility (e.g., Hospital, Private Practice, Clinic) where the study took place. Actual and projected estimates of the number of AK eyes in years were calculated multiplying the ratio of AK to the total population and the corresponding present and projected population estimates (age range: 15 to 70), sourced from the United Nations (UN) Population Prospects.
RESULTS: Overall, 105 articles were included, published between 1987 and 2022. The total number of eyes identified was 91,951, with 5,660 affected by AK and 86,291 by non-viral MK. The median ACI was 1.9 new AK eyes per healthcare center per year (95%CI of the median: 1.5 to 2.6), with no statistically significant differences observed among continents. The ratio of AK eyes to the total number of MK eyes was 1.52% (95%CI: 1.02% to 2.24%), while the ratio of AK in relation to the entire population was estimated at 0.0002% (95%CI: 0.0001 to 0.0006), or 2.34 eyes per 1,000,000 subjects (95%CI: 0.98 to 5.55 per 1.000.000 subjects). The projected increase in the numbers of AK eyes indicates a rise of +18.5% (15,356 AK eyes) in 2053 and +25.5% (16,253 AK eyes) in 2073, compared to the baseline of 2023 (12,954 AK eyes) CONCLUSION: AK emerged as a relatively low-incident disorder, and no significant differences in terms of its incidence were found among different continents.