Reliable and specific detection of Acanthamoeba spp. in dishcloths using quantitative real-time PCR assay.

Acanthamoeba spp., are ubiquitous protist which belongs to Free-Living Amoeba (FLA) group, is considered as causal agent of side-threatening keratitis or fatal encephalitis among other human infections. Besides, this parasite has been reported as host for other microorganisms important to human health such as Campylobacter spp. or Vibrio spp. among others. This role of Acanthamoeba as pathogen and environmental phagocyte has increased the reports confirming its presence in human related environments, acting as a water quality indicator. Considering the tide relationship between water and kitchen environments, and the high prevalence of Acanthamoeba in water sources, the present study aims to establish a quick and accurate protocol based on DNA extraction and a real time qPCR assay to detect Acanthamoeba spp. in dishcloths. The procedure has been validated by processing 17 used dishcloths. Our findings demonstrated the high sensitivity of the qPCR assay used which was capable of detecting up to one Acanthamoeba from an in vitro contaminated dishcloth. The protocol accurately detected 64.7% of positive samples for Acanthamoeba spp, (in 4 samples DNA concentrations corresponded to 1-102 amoebae). Our findings demonstrate the importance of FLA surveillance by efficient and sensitive methods since one amoeba is capable of colonizing human related food environments such as kitchens sinks and could be a potential source of infection.

Presence and diversity of free-living amoebae and their potential application as water quality indicators.

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.

Adhesion of Acanthamoeba on Scleral Contact Lenses According to Lens Shape.

Purpose: To investigate the adhesion of Acanthamoeba to scleral contact lens (ScCL) surface according to lens shape. Methods: Two strains of A. polyphaga (CDC:V062 and ATCC 30461) and one clinical Acanthamoeba isolate, were inoculated onto five contact lens (CL): one first-generation silicone hydrogel (SHCL; lotrafilcon B; adhesion control) containing plasma surface treatment; two ScCL (fluorosilicone acrylate) one containing surface treatment composed of plasma and the other containing plasma with Hydra-PEG, and two CL designed with a flat shape having the same material and surface treatments of the ScCL. Trophozoites that adhered to the lens's surfaces were counted by inverted optical light microscopy. Possible alterations of the lens surface that could predispose amoeba adhesion and Acanthamoeba attached to these lens surfaces were evaluated by scanning electron microscopy (SEM). Results: All strains revealed greater adhesion to the ScCL when compared with the flat lenses (P < 0.001). The clinical isolate and the ATCC 30461 had a higher adhesion (P < 0.001) when compared with the CDC:V062. A rough texture was observed on the surface of the lenses that have been examined by SEM. Also, SEM revealed that the isolates had a rounded appearance on the surface of the ScCL in contrast with an elongated appearance on the surface of the silicone hydrogel. Conclusions: The findings revealed that the curved shape of the ScCL favors amoeba adhesion.

Diagnostic utility of in vivo confocal microscopy in Acanthamoeba keratitis following corneal crosslinking.

Acanthamoeba keratitis (AK) is a rare but potentially sight-threatening complication of corneal collagen crosslinking (CXL) for keratoconus. In this report, we describe an early adolescent male who underwent routine CXL for progressive keratoconus in his left eye. Preprocedural left visual acuity (VA) was 6/9. At day 5 postprocedure, multifocal corneal infiltrates were identified. Corneal scrape, bandage contact lens cultures and herpetic and Acanthamoeba PCR were negative. In vivo, confocal microscopy (IVCM) identified Acanthamoeba cysts within the corneal stroma. Intensive amoebicidal therapy was initiated, but recovery was complicated by significant inflammation, resulting in widespread aggressive corneal vascularisation necessitating topical steroids and steroid-sparing agents. At 10 months, his left VA was 6/24. This report emphasises the importance of maintaining a high index of suspicion for AK in cases of post-CXL microbial keratitis and highlights the diagnostic value of IVCM, particularly in culture-negative and PCR-negative cases.

Microbial keratitis in Southern Malawi: a microbiological pilot study.

OBJECTIVE: Microbial keratitis (MK) is a significant cause of blindness in sub-Saharan Africa. We investigated the feasibility of using a novel corneal impression membrane (CIM) for obtaining and processing samples by culture, PCR and whole-genome sequencing (WGS) in patients presenting with suspected MK in Malawi. METHODS AND ANALYSIS: Samples were collected from patients presenting with suspected MK using a 12 mm diameter polytetrafluoroethylene CIM disc. Samples were processed using culture and PCR for Acanthamoeba, herpes simplex virus type 1 (HSV-1) and the bacterial 16S rRNA gene. Minimum inhibitory concentrations of isolates to eight antimicrobials were measured using susceptibility strips. WGS was used to characterise Staphylococcus aureus isolates. RESULTS: 71 eyes of 71 patients were included. The overall CIM isolation rate was 81.7% (58 positive samples from 71 participants). 69 (81.2%) of isolates were Gram-positive cocci. Coagulase-negative Staphylococcus 31.8% and Streptococcus species 14.1% were the most isolated bacteria. Seven (9.9%) participants were positive for HSV-1. Fungi and Acanthamoeba were not detected. Moxifloxacin and chloramphenicol offered the best coverage for both Gram-positive and Gram-negative isolates when susceptibility was determined using known antimicrobial first quartile concentrations and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively. WGS identified known virulence genes associated with S. aureus keratitis. CONCLUSIONS: In a resource-poor setting, a CIM can be used to safely sample the cornea in patients presenting with suspected MK, enabling identification of causative microorganisms by culture and PCR. Although the microbiological spectrum found was limited to the dry season, these preliminary results could be used to guide empirical treatment.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Chitosan nanoparticles improve the effectivity of miltefosine against Acanthamoeba.

BACKGROUND: Acanthamoeba keratitis (AK) is a corneal sight-threatening infection caused by the free-living amoebae of the genus Acanthamoeba. Early and appropriate treatment significantly impacts visual outcomes. Mucoadhesive polymers such as chitosan are a potential strategy to prolong the residence time and bioavailability of the encapsulated drugs in the cornea. Regarding the recent administration of miltefosine (MF) for treating resistant AK, in the present study, we synthesized miltefosine-loaded chitosan nanoparticles (MF-CS-NPs) and evaluated them against Acanthamoeba. METHODOLOGY/PRINCIPAL FINDINGS: Chitosan nanoparticles (CNPs) were prepared using the ionic gelation method with negatively charged tripolyphosphate (TPP). The zeta-potential (ZP) and the particle size of MF-CS-NPs were 21.8±3.2 mV and 46.61±18.16 nm, respectively. The release profile of MF-CS-NPs indicated linearity with sustained drug release. The cytotoxicity of MF-CS-NPs on the Vero cell line was 2.67 and 1.64 times lower than free MF at 24 and 48 hours. This formulation exhibited no hemolytic activity in vitro and ocular irritation in rabbit eyes. The IC50 of MF-CS-NPs showed a significant reduction by 2.06 and 1.69-fold in trophozoites at 24 and 48 hours compared to free MF. Also, the MF-CS-NPs IC50 in the cysts form was slightly decreased by 1.26 and 1.21-fold at 24 and 48 hours compared to free MF. CONCLUSIONS: The MF-CS-NPs were more effective against the trophozoites and cysts than free MF. The nano-chitosan formulation was more effective on trophozoites than the cysts form. MF-CS-NPs reduced toxicity and improved the amoebicidal effect of MF. Nano-chitosan could be an ideal carrier that decreases the cytotoxicity of miltefosine. Further analysis in animal settings is needed to evaluate this nano-formulation for clinical ocular drug delivery.

Global prevalence of potentially pathogenic free-living amoebae in sewage and sewage-related environments-systematic review with meta-analysis.

Free-living amoebae (FLA) include amphizoic microorganisms important in public health, widely isolated from air, water, and soil. However, its occurrence in sewage-related environments still needs to be systematically documented. This study summarizes the occurrence of FLA in sewage-related environments through a systematic review with meta-analysis. A total of 1983 scientific article were retrieved from different databases, of which 35 were selected and analyzed using a random effects forest plot model with a 95% confidence interval (IC). The pooled overall prevalence of FLA in sewage across 12 countries was 68.96% (95% IC = 58.5-79.42). Subgroup analysis indicates high prevalence in all environments analyzed, including sewage water from the sewage treatment plant (81.19%), treated sewage water (75.57%), sewage-contaminated water (67.70%), sediment contaminated by sewage (48.91%), and sewage water (47.84%). Prevalence values of Acanthamoeba spp., Hartmanella/Vermamoeba spp., and Naegleria spp. are 47.48%, 28.24%, and 16.69%, respectively. Analyzing the species level, the distribution is as follows: Acanthamoeba palestinensis (88%), A. castellanii (23.74%), A. astronyxis (19.18%), A. polyphaga (13.59%), A. culbertsoni (12.5%), A. stevensoni (8.33%), A. tubiashi (4.35%) and A. hatchetti (1.1%), Naegleria fowleri (28.4%), N. gruberi (25%), N. clarki (8.33%), N. australiensis (4.89%) and N. italica (4.29%), Hartmannella/Vermamoeba exundans (40%) and H.V. vermiform (32.61%). Overall, our findings indicate a high risk associated with sewage-related environments, as the prevalence of FLA, including pathogenic strains, is high, even in treated sewage water. The findings of this study may be valuable both for risk remediation actions against amoebic infections and for future research endeavors.

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.

Molecular detection and characterization of Acanthamoeba infection in dogs and its association with keratitis in Korea.

Acanthamoeba infection is associated with keratitis in humans; however, its association with keratitis in dogs remains unclear. To investigate this possibility, we collected 171 conjunctival swab samples from dogs with eye-related diseases (65 with keratitis and 106 without keratitis) at Chungbuk National University Veterinary Teaching Hospital, Korea, from August 2021 to September 2022. Polymerase chain reaction identified 9 samples (5.3%) as Acanthamoeba positive; of these, 3 were from dogs with keratitis (4.6%) and 6 were from dogs without keratitis (5.7%). Our results indicated no significant association between Acanthamoeba infection and keratitis, season, sex, or age. All Acanthamoeba organisms found in this study had the genotype T4, according to 18S ribosomal RNA analysis. Acanthamoeba infection in dogs might have only a limited association with keratitis.

Are Thermotolerant and Osmotolerant Characteristics of Acanthamoeba Species an Indicator of Pathogenicity?

Objective: The aim of this study was to evaluate the pathogenicity of Acanthamoeba strains with T4, T5, T11, and T12 genotypes by comparing the osmotolerance and thermotolerance characteristics of Acanthamoeba strains isolated from genotype groups, within species with the same genotype, and from environmental and keratitis cases. Methods: In this study, after axenic cultures of 22 Acanthamoeba strains with T4 (Neff, A, B, D, E), T5, T11, and T12 genotypes isolated from clinical and environmental samples, thermotolerance (37 °C, 39 °C and 41 °C) and osmotolerance (0.5 M, 1 M) tests were performed. Results: All strains showed growth ability at 37 °C and 0.5 M osmolarity. While all five strains isolated from patients with Acanthamoeba keratitis showed growth ability at 37 °C and 0.5 M osmolarity, no growth was detected at 41 °C and 1 M osmolarity. When the tolerance characteristics of the strains with the same genotype were evaluated, the strains with the T5 and T4E genotypes showed the same characteristics. When Acanthamoeba strains with the T4 genotype were evaluated in general, 31.25% of the strains were found to grow at 39 °C and 6.25% at 41 °C. Of the T4Neff strains, only one strain did not show the ability to reproduce at 39 °C and showed a different feature from the other strains. While the strain with the T11 genotype grew at all temperatures, the strain with the T12 genotype did not grow at 41 °C. Conclusion: According to our research results, we believe that tolerance to 39 °C and 1 M mannitol is not an indicator of pathogenicity. More studies with Acanthamoeba strains are required to clarify this issue.

Molecular identification, phylogenetic analysis and histopathological study of pathogenic free-living amoebae isolated from discus fish (Symphysodon aequifasciatus) in Iran: 2020-2022.

Free-living amoebae (FLA) are capable of inhabiting diverse reservoirs independently, without relying on a host organism, hence their designation as "free-living". The majority of amoebae that infect freshwater or marine fish are amphizoic, or free-living forms that may colonize fish under particular circumstances. Symphysodon aequifasciatus, commonly referred to as the discus, is widely recognized as a popular ornamental fish species. The primary objective of the present study was to determine the presence of pathogenic free-living amoebae (FLA) in samples of discus fish. Fish exhibiting clinical signs, sourced from various fish farms, were transferred to the ornamental fish clinic. The skin, gills, and intestinal mucosa of the fish were collected and subjected to culturing on plates containing a 1% non-nutrient agar medium. The detection of FLA was conducted through morphological, histopathological and molecular methods. The construction of the phylogenetic tree for Acanthamoeba genotypes was achieved using the maximum likelihood approach. The molecular sequence analysis revealed that all cultures that tested positive for FLA were T4 genotype of Acanthamoeba and Acanthamoeba sp. The examination of gill samples using histopathological methods demonstrated the presence of lamellar epithelial hyperplasia, significant fusion of secondary lamellae, and infiltration of inflammatory cells. A multitude of cysts, varying in shape from circular to elliptical, were observed within the gills. The occurrence of interlamellar vesicles and amoeboid organisms could be observed within the epithelial tissue of the gills. In the current study, presence of the Acanthamoeba T4 genotype on the skin and gills of discus fish exhibiting signs of illness in freshwater ornamental fish farms was identified. This observation suggests the potential of a transmission of amoebic infection from ornamental fish to humans, thereby highlighting the need for further investigation into this infection among ornamental fish maintained as pets, as well as individuals who interact with them and their environment.

Clinical outcomes and prognostic factors in Acanthamoeba keratitis.

PURPOSE: To report clinical findings and prognostic factors for visual and morphological outcomes in patients with Acanthamoeba keratitis (AK). METHODS: Single-center, retrospective, longitudinal study of 51 cases of AK diagnosed by real-time polymerase chain reaction (RT-PCR) between March 2010 and October 2022. The primary outcome was the final best corrected visual acuity (BCVA). Poor visual outcome was defined as a final BCVA ≥ 1 logMAR unit, while good visual outcome was defined as a final BCVA < 1 logMAR unit. Eyes from these two groups were compared, regarding demographic and initial clinical variables, anti-Acanthamoeba treatment used, and complications of the disease. Early diagnosis was defined as ≤ 14 days from symptom onset to diagnostic confirmation and initiation of Acanthamoeba medical treatment. Multivariable logistic regression was used to determine predictors of poor visual outcome. RESULTS: A total of 51 eyes from 46 patients diagnosed with AK, all contact lens (CL) wearers, were included in this study. Average follow-up was 39.0 ± 30.2 [total range 14-120] months. Thirty-one eyes (60.8 %) presented good visual outcome, with a lower baseline age (30.5 ± 9.0 vs. 42.3 ± 15.8; p = 0.020), better initial BCVA (0.8 ± 0.7 logMAR units vs. 1.3 ± 0.9 logMAR units; p = 0.047), higher rate of early diagnosis (45.2 % vs. 5.6 %; p = 0.004), and higher rate of therapeutic epithelial debridement (64.5 % vs. 10 %; p < 0.001). 20 eyes (39.2 %) presented poor visual outcome, with 12 eyes undergoing evisceration/enucleation (23.5 %). These 20 eyes presented a higher rate of complications (90 % vs. 61.3 %; p = 0.031). In multivariable analysis, early diagnosis of AK (OR 19.78; 95 % CI 2.07-189.11; p = 0.010) and therapeutic epithelial debridement (OR 19.02; 95 % CI 3.27-110.57; p = 0.001) were associated with a good visual outcome. CONCLUSIONS: In the present study, poor visual outcome was present in 39 % of affected eyes. Early AK diagnosis (≤14 days from symptom onset) and therapeutic epithelial debridement were associated with good final visual outcome.

First Report of Isolation and Characterization of Acanthamoeba spp. from the Milk Used for Calf Feeding.

PURPOSE: Acanthamoeba spp. can be found in natural and artificial environments, which reflects their high adaptability to different conditions. Based on the available data, there is scarce information about the isolation of amoeba from milk. This study aimed to investigate the probable presence of Acanthamoeba in milk used for calf feeding. METHODS: 200 milk samples from 50 industrial and traditional farms were collected. The samples were filtered and cultured on the 1.5% Non-nutrient agar medium. The amoebic growth was examined with an inverted microscope daily. DNA was extracted from the positive plates, and a PCR reaction was undertaken using the primers amplifying the Acanthamoeba 18 S rRNA gene. Five samples were purified and sequenced using specific primers. Maximum likelihood reconstructions were performed using the phylogenetic program MEGA software. The osmo and thermotolerance of isolated trophozoites were examined as well. RESULTS: Out of 200 milk samples, Acanthamoeba was isolated from 27 (13.5%). The phylogenetic tree represents that all the isolates belonged to the genotype T4. Results of thermo and osmotolerance tests showed that isolates could develop at 37 and 43 ◦C. Besides, trophozoites survived at 0.5 M mannitol and 1 M. CONCLUSION: For the first time, Acanthamoeba spp. were isolated from milk used to feed dairy calves. Due to Acanthamoeba's neglected role in pathogen persistence and survival, hygiene instructions should be reconsidered.

Phylogenetic analysis of Acanthamoeba isolated from soil samples and nasal cavities of patients with malignancy: a public health concern in the northwest of Iran.

BACKGROUND: The genus Acanthamoeba is reported from various environmental sources and can cause multiple complications, including chronic amoebic aeratitis and amoebic granulomatous encephalitis. This study investigated the presence and genotyping of Acanthamoeba in the soil of parks and patients with malignancies referred to health centers in Zanjan city, Iran. METHODS: In this cross-sectional study, 200 soil samples were collected from amusement parks in Zanjan city from September 2017 to May 2018. Samples were cultured on 1.5% non-nutrient agar, and the Acanthamoeba genus was identified using the morphological method. PCR was performed on all positive environmental samples, and six microscopically positive clinical samples belonged to our previous study. DNA sequencing of 18S rRNA was performed to analyze the genetic pattern of some PCR-positive isolates. RESULTS: Microscopic results showed that 96 (48%) soil samples were positive. PCR confirmed all positive cases of clinical samples and 84 soil samples. Out of the PCR-positive samples, 20 soil samples and five clinical samples were sequenced successfully. All soil isolates belonged to the T4 genotype, and three and two clinical samples belonged to T4 and T5 genotypes, respectively. CONCLUSION: : The presence of Acanthamoeba in both the environment and clinical samples of Zanjan city suggests paying greater attention to the infections caused by it.

The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model.

BACKGROUND: Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. METHODOLOGY/PRINCIPAL FINDINGS: Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne-functionalized D-alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P. aeruginosa which was seen throughout the Acanthamoeba's cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 (p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. CONCLUSIONS/SIGNIFICANCE: Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis.

Case Report: A Series of Three Meningoencephalitis Cases Caused by Acanthamoeba spp. from Eastern India.

Acanthamoeba spp. are rare etiological agents of meningoencephalitis with high mortality. We present three cases of Acanthamoeba meningoencephalitis in immunocompetent individuals from Eastern India. The first patient presented with fever and headache; the second with headache, visual disturbance, and squint; and the third presented in a drowsy state. The cases presented on March 3, 18, and 21, 2023 respectively. The first two patients had concomitant tubercular meningitis for which they received antitubercular therapy and steroid. Their cerebrospinal fluid showed slight lymphocytic pleocytosis and increased protein. The diagnosis was done by microscopy, culture, and polymerase chain reaction. They received a combination therapy comprising rifampicin, fluconazole, and trimethoprim-sulfamethoxazole. The first patient additionally received miltefosine. She responded well to therapy and survived, but the other two patients died despite intensive care. Detection of three cases within a period of 1 month from Eastern India is unusual. It is imperative to sensitize healthcare providers about Acanthamoeba meningoencephalitis to facilitate timely diagnosis and treatment of the disease.

A brief history of metal recruitment in protozoan predation.

Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth's history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth's history. The origin of phagotrophic eukaryotes must have postdated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later - closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.

Diagnosing and monitoring the characteristics of Acanthamoeba keratitis using slit scanning and laser scanning in vivo confocal microscopy.

INTRODUCTION: Acanthamoeba keratitis (AK) is a serious and potentially blinding ocular infection caused by the free-living amoeba, Acanthamoeba. In vivo confocal microscopy (IVCM) is a non-invasive device which has been proven of great use to diagnose Acanthamoeba infections immediately. The aim of this review was to establish different patterns and signs of AK that appear on the IVCM both before and after treatment. METHODS: A systematic review of the literature from 1974 until September 2021 was performed using Embase and PubMed, following The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: Twenty different signs of AK were observed using IVCM. The included studies used vastly different criteria to diagnose infections, ranging from just 1 to 13 of the signs, demonstrating the current lack of a standardised diagnosis of this infection using the IVCM. The appearance of double wall cysts, trophozoites, signet rings, target signs and clusters were shown to be pathognomonic to AK infections. Bright spots located in the corneal epithelium were demonstrated as non-reliable predictors of AK. The presence of cysts in clusters and single file can predict the need for corneal transplantation. The morphological changes in cysts using the IVCM following treatment were described as breaking down to hollow forms and occasionally surrounded by black cavities. Using this information, a visual guideline for identifying AK signs in diagnosis and follow-up using IVCM was created. CONCLUSION: Increased awareness of the different signs and patterns of AK that appear on the IVCM is crucial in order to correctly identify an infection and increase the potential of this device. Our guidelines presented here can be used, but further studies are needed in order to determine the relationship and aetiology of these signs and cellular changes on the IVCM both before and after anti-amoeba treatment.