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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. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis is a rare parasitic infection of the cornea that can lead to permanent blindness if not diagnosed and treated promptly. We collected data on the incidences of Acanthamoeba keratitis from 20 countries and calculated an annual incidence of 23,561 cases, with the lowest rates in Tunisia and Belgium, and the highest in India. We analyzed 3755 Acanthamoeba sequences from the GenBank database across Asia, Europe, North America, South America, and Oceania and genotyped them into T1, T2, T3, T4, T5, T10, T11, T12, and T15. Many genotypes possess different characteristics, yet T4 is the most prevalent genotype. As efficient treatment against Acanthamoeba remains lacking, prevention from early diagnosis via staining, PCR, or in vivo confocal microscopy (IVCM) becomes significant for the condition's prognosis. IVCM is the most recommended approach for the early detection of Acanthamoeba. If IVCM is unavailable, PCR should be used as an alternative. [ABSTRACT FROM AUTHOR]

Contact lens care and early diagnosis of Acanthamoeba keratitis (AK) are very important to prevent progression to blindness due to AK, which develops when Acanthamoeba attaches to contact lens-damaged corneas. Therefore, we propose a novel, non-invasive, immuno-surface-enhanced Raman scattering (SERS) sensing platform for rapid and accurate detection of Acanthamoeba infection in the tears and contact lens solutions of humans. This optic analysis method was based on the proven biological performance of chorismate mutase (CM) - specific monoclonal and polyclonal antibodies on trophozoite and cyst forms of Acanthamoeba castellanii, and its conditioned media. SERS-based, ultra-low concentration detection was achieved by the anisotropic fanblade-shaped core-shell nanoassembly (Ag@AuFNP) embedded with 4-fluorobenzenethiol Raman reporter. The immuno-SERS platform combining Ag@AuFNP and CM-specific antibody complexes was evaluated in vitro and in vivo. The non-invasive SERS-activated biosensing platform indicates strong feasibility for AK detection in human tears and contact lens solutions. [Display omitted] • The SERS immunoassay nanoplatform was first proposed for early detection of Acanthamoeba keratitis. • Nanofanblade-shaped anisotropic core-shell nanoassemblies were proposed for highly sensitive detection. • It was implemented through the fusion of a validated single-clone antibody for CM and immuno-SERS. • It provided rapid and accurate detection of Acanthamoeba infections in tears and contact lens solutions of humans. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics. [ABSTRACT FROM AUTHOR]

Background: Acanthamoeba keratitis (AK) is a vision-threatening disease, usually associated with contact lens (CL) wear. As overnight orthokeratology (OOK) is increasingly used to control myopia, we have found incidence of OOK-associated AK is increasing. This study aimed to investigate the clinical presentation and visual outcomes of OOK-associated AK. Methods: Demographic characteristics, clinical features, and treatment outcomes were collected by reviewing the medical charts of CL-associated AK patients (n = 35) diagnosed at Taipei Veterans General Hospital from 2001 to 2016. Cases were OOKassociated AK patients (n = 13), and controls were all other CL-associated AK patients (n = 22). Student t tests and chi-square tests were used to compare cases and controls. Linear regression analyses were used to identify factors associated with the final visual outcome in CL-associated AK. Results: OOK-associated AK accounted for half of all CL-associated AK after 2010. OOK-associated AK patients and other CL-associated patients had similar best-corrected logarithm of the minimum angle of resolution visual acuity (BCLVA) before treatment (1.10 ± 0.75 vs 1.13 ± 0.76, p = 0.893), but OOK-associated AK patients were younger (17.15 ± 3.21 vs 26.36 ± 12.81 years, p = 0.004), had less severe disease (ring infiltration, 0% vs 31.82%, p = 0.023), and had better post-treatment BCLVA (0.06 ± 0.15 vs 0.51 ± 0.95, p = 0.041). Multiple linear regression analysis showed that better BCLVA after treatment in CL-associated AK was associated with initial presentation without ring infiltration (p = 0.002) but not with OOK use itself (p = 0.793). Twenty-six of 35 CL-associated AK patients had final BCLVA equal to or better than 0.10 (Snellen visual acuity of 6/7.5). All 13 OOK-associated AK cases were treated with chlorhexidine 0.02% ± voriconazole 1% ± oral voriconazole, and 12 of these patients had final BCLVA equal to or better than 0.10. Conclusion: Most CL-associated AK patients had satisfactory visual outcomes. Half of AK at our hospital is OOK-associated since 2010. Early diagnosis and correct treatment may be the reason why OOK-associated AK patients had better vision prognosis. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellaniispecific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK), the vision-threatening disease caused by the amphizoic, potentially parasitic amoebae is growing threat for public health in Poland and worldwide. The report presents the case of 70-year-old man with severe keratitis admitted to an Ophthalmology Clinic. Before admission, the patient had been treated for 6 months with antibacterial and antifungal drugs in other units, without improvement in the eye condition. The use of in vivo confocal microscopy and in vitro cultivation allowed diagnosis to be verified and AK successfully treated. Awareness of the threat to public health caused by Acanthamoeba spp is still insufficient. If there is failure in response to first line therapy, AK should be taken into account,despite the lack of identified risk factors. In vitro monitoring of amoebic strain can be helpful for prognosis of the course of the corneal disease. Improvement in duration from first symptoms until proper diagnosis is decisive for better treatment efficacy. [ABSTRACT FROM AUTHOR]

Copyright of Arquivos Brasileiros de Oftalmologia is the property of Arquivos Brasileiros de Oftalmologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Acanthamoeba castellanii , a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3′-nucleotidase/nuclease (3′-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii , are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3′-AMP, and this activity is similar to that observed in other protists. The addition of 3′-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3′-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3′-AMP. We believe that ecto-3′-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3′-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells. [ABSTRACT FROM AUTHOR]

Introduction: The usage of contact lens among the community is increasing due to trend and as an alternative of wearing glasses. However, most ocular diseases and eye infections such as Acanthamoeba keratitis are more related to contact lens usage. The contact lens disinfecting solutions (CLDS) are supposed to be able to eradicate the microorganisms that could possibly cause eye infections. Objective: This study was conducted to determine the effectiveness of the contact lens disinfecting solutions against Acanthamoeba spp. cysts of clinical isolates based on manufacturer's recommended soaking time and variable soaking times. Materials and Methods: CLDS were tested against two isolates of Acanthamoeba spp. in durations recommended by the product manufacturer; 4 and 6 hours and also in extended duration of 8 hours soaking. The disinfecting solutions tested were AOSept Plus and Biotrue® multipurpose solution against two clinical isolates (HKL 55 & HS 19). The Acanthamoeba spp. isolates were subcultured onto non-nutrient agar and the cysts produced were mixed with disinfectants separately according to different soaking hours. After the soaking duration, the cysts were cultured onto non-nutrient agar layered with heat-killed E. coli and incubated at 30°C. The cultures were observed under inverted microscope for 14 days continuously to see any excystment of the cyst. The failure of the cysts to excyst indicates the efficiency of the disinfectants to kill the cyst successfully. Results: AOSept Plus exhibited good anti-Acanthamoeba activity and was able to inhibit the excystment of Acanthamoeba spp. cysts in all isolates. Biotrue® Multipurpose solution failed to exhibit anti-Acanthamoeba activity. Conclusion: AOSept Plus representing the hydrogen peroxide based contact lens disinfecting solution is a choice for contact lens wearers if Acanthamoeba keratitis is to be prevented. [ABSTRACT FROM AUTHOR]

Purpose: To study the surgical outcome of therapeutic penetrating keratoplasty (TPK) for medically uncontrolled microbial keratitis at a tertiary hospital in Taiwan over a 14-year period.Methods: This is a retrospective case series study. Patients who underwent TPK at National Taiwan University Hospital in 2001-2014 were included. Patients were divided into 3 diagnostic groups: bacterial keratitis, fungal keratitis, and acanthamoeba keratitis. Each of the following criteria was evaluated: graft clarity at 1 month and 1 year postoperatively, cure of the disease, and anatomical success rate.Results: A total of 107 TPKs were included. TPK eradicated the infection in 57/62 (91.9%) of bacterial keratitis, 33/41 (80.5%) of fungal keratitis, and 9/10 (90.0%) of acanthamoeba keratitis. 22/57 grafts (38.6%) of bacterial keratitis, 22/38 grafts (57.9%) of fungal keratitis, and 5/10 grafts (50.0%) of acanthamoeba keratitis remained clear at 1 year postoperatively. The 1-year graft survival rate did not significantly differ among these 3 groups. The leading causes of graft failure were late endothelial decompensation and graft reinfection. A higher percentage of graft clarity was achieved in grafts <8.5 mm in diameter than in larger grafts (55.4% vs. 31.4%, P = 0.027). Of the 14 patients with endophthalmitis at the time of TPK, the infection was successfully treated in 13 patients except 1 patient required evisceration.Conclusion: TPK is valuable in the management of medically uncontrolled microbial keratitis, although the 1-year graft survival rate is unsatisfactory. TPK combined with intravitreal antibiotics and vitrectomy is also a beneficial treatment strategy for patients with endophthalmitis secondary to microbial keratitis. [ABSTRACT FROM AUTHOR]

• Intrastromal Injection can be considered in deep Acanthamoeba keratitis (AK). • Intrastromal injection of 0.01% PHMB or 0.05% propamidine isethionate is safe. • This model system could help to determine the toxic effect of other agents. • Further experiments may determine the toxicity of multiple intrastromal injection. • AK animal model is required to evaluate the true effect of intrastromal injection. Acanthamoeba keratitis (AK) is difficult to treat, especially when the corneal deep stroma is involved. Intrastromal injection of antimicrobial agents is an effective adjuvant therapy for deep recalcitrant microbial keratitis; however, it has not been used to treat AK due to suspected drug toxicity. The purpose of this study was to evaluate the toxicity of corneal intrastromal injection of polyhexamethylene biguanide (PHMB) and propamidine isethionate (Brolene®, Sanofi) in New Zealand white rabbits. We performed intrastromal injections of PHMB (0.02 or 0.01%) and propamidine isethionate (0.1 or 0.05%) into the rabbits' right corneas. The left corneas were injected with phosphate-buffered saline as controls. The rabbits were sacrificed on the 7th day after injection, and the corneal buttons were harvested for further evaluation by slit lamp microscopy, specular microscopy, hematoxylin and eosin staining, scanning electron microscopy, terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling assays, and WST-1 assays. We found that intrastromal injection of 0.02% PHMB or 0.1% propamidine isethionate resulted in corneal epithelial erosion, corneal edema, and severe neovascularization. However, 0.01% PHMB or 0.05% propamidine isethionate did not induce obvious cornea toxicity. In conclusion, intrastromal injection of 0.01% PHMB or 0.05% propamidine isethionate may be promising adjunctive treatments for deep stromal AK. [ABSTRACT FROM AUTHOR]

To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period. This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes. Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p = 0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83 mm, p = 0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p = 0.02) and dilated pupil/iris atrophy (p = 0.01). Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma. [ABSTRACT FROM AUTHOR]

Purpose: To analyze the outcomes of Acanthamoeba keratitis (AK) in terms of different clinical presentations in a tertiary hospital in Taiwan over a 20- year period.Methods: This is a retrospective case series. Patients with AK diagnosed at the National Taiwan University Hospital between January 1996 and December 2015 were identified. A diagnosis of AK was made on the basis of positive Acanthamoeba smear/cultures or pathological identification of Acanthamoeba cysts on keratoplasty specimens. Patient demographics, clinical presentations, treatment courses, and final visual outcomes were collected and analyzed. Visual acuity, postoperative complications and graft survivals were measured as outcomes.Results: Of the 62 patients with AK in our study, 64.5% were medically treated while 35.5% underwent surgical treatment. In those with ring infiltrate, 52.4% patients could be successfully treated with medications. In eyes receiving penetrating keratoplasty, postoperative complications were more common in therapeutic penetrating keratoplasty (TPK) than those in optical penetrating keratoplasty (OPK) group (82.4% versus 40%, p = 0.04). The graft size was larger in TPK than that in OPK group (8.56 versus 7.83 mm, p = 0.002). Furthermore, post-operative glaucoma, which was the major complication, was found to be associated with larger graft size (p = 0.02) and dilated pupil/iris atrophy (p = 0.01).Conclusion: Even in advanced cases with ring infiltrate, eradication of infection with anti-amoebic drugs is possible. In those requiring keratoplasty, the surgical timing should be made meticulously considering graft size and signs of dilated pupil/iris atrophy in terms of post-operative glaucoma. [ABSTRACT FROM AUTHOR]

Introduction: Increasing numbers of Acanthamoeba keratitis (AK) cases are reported from many countries including Malaysia, in line with the popularity contact lens wear. However, the true extent or exact frequency of the disease is not known in Malaysia since it is not a notifiable disease in this country. Objective: This study aims to retrospectively analyze all cases diagnosed at Hospital Universiti Kebangsaan Malaysia (HUKM) in a 3-year period between 2010-2012. Materials and Methods: This retrospective study involved clinical samples submitted to the Acanthamoeba laboratory at the Department of Parasitology, HUKM from the year 2010-2012. The study only included patients diagnosed as having microbial keratitis with Acanthamoeba as the presumptive causative agent. Records from the request forms for Acanthamoeba isolation and the culture results were analyzed. Results: A total of 64 suspected cases had their corneal scrapings sent to the Acanthamoeba laboratory from various hospitals across Malaysia. 10 (15.6%) parasitologically confirmed culture positive cases were identified from the total 64 suspected cases during the period 2010-2012. All 10 positive cases were contact lens wearers and were predominantly women (80%) while men (20%). All patients presented with similar symptoms of redness, pain, watery eye and photophobia. Conclusion: All 10 AK cases were diagnosed at only one study center and it would be anticipated that the number of cases would increase if there were more study centers involved throughout Malaysia. This study has proven that AK is not extremely rare in Malaysia and it should be considered as part of the differential diagnosis of most cases of presumed microbial keratitis especially those involving contact lens wearers. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a rare sight-threatening corneal infection, often reporting from contact lens wearers. An asymptomatic human immunodeficiency virus (HIV)-infected Thai male without history of contact lens use complained foreign body sensation at his left eye during motorbike riding. He had neither specific keratitis symptoms nor common drugs responding, which contributed to delayed diagnosis. By corneal re-scraping, Acanthamoeba-like cysts were detected by calcofluor white staining and agar culture. The etiological agent obtained from the culture was molecularly confirmed by Acanthamoeba spp.-specific PCR, followed by DNA sequencing. The results from BLAST and phylogenetic analysis based on the DNA sequences, revealed that the pathogen was Acanthamoeba T4, the major genotype most frequently reported from clinical isolates. The infection was successfully treated with polyhexamethylene biguanide resulting in corneal scar. This appears the first reported AK case from a non-contact lens wearer with HIV infection in Thailand. Although AK is sporadic in developing countries, a role of free-living Acanthamoeba as an opportunistic pathogen should not be neglected. The report would increase awareness of AK, especially in the case presenting unspecific keratitis symptoms without clinical response to empirical antimicrobial therapy. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis, a vision-threatening disease, is caused by a free-living amoeba of genus Acanthamoeba. Amoeba is widely distributed all around the world. Several cases of Acanthamoeba keratitis have been reported from the Middle East and Iran as well. The infection is mainly caused by inappropriate use of contact lens; therefore, it is more common among lens wearers. The number of reported cases worldwide is increasing annually due to the increasing number of contact lens wearers for medical or cosmetic reasons. It is known that early diagnosis and treatment can reduce consequent damages, while the delay in these processes will deteriorate the vision. Nowadays contact lens wearing, surgery and use of corticosteroids are known to predispose to Acanthamoeba keratitis, and it was also believed that trauma was the main cause. Increasing public knowledge about Acanthamoeba infection, inquiring the history of patients, clinical signs and laboratory findings can be helpful to early diagnosis and better treatment. Regardless of the increasing knowledge of diagnosis and treatment, this disease is still a challenge. Considering the long curative time, and insufficient efficacy of available treatments, it seems that the prevention is more important than the treatment. This review aimed to explain Acanthamoeba keratitis in the Middle East and Iran from the aspects of epidemiology, diagnosis, and therapeutic treatment. [ABSTRACT FROM AUTHOR]

Copyright of Arquivos Brasileiros de Oftalmologia is the property of Arquivos Brasileiros de Oftalmologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Introduction. Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease - Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. Materials and method. Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25-2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. Results. Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. Conclusions. Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users. [ABSTRACT FROM AUTHOR]

Introduction: Keratitis is one of the common causes of visually impaired cases in Malaysia and Acanthamoeba is known to cause severe keratitis leading to blindness if left untreated. Contact lens wear is a major risk factor for Acanthamoeba keratitis (AK) and therefore effective contact lens disinfectants are important in preventing the infection. Objective: This study was performed to evaluate the anti-Acanthamoeba activity of a locally produced commercial contact lens disinfecting solution (CLDS), OPTICARE Multimate®, towards the cysts of Acanthamoeba isolates. Materials and Methods: Two environmental isolates of Acanthamoeba; TTT1 & TKT19 were exposed to OPTICARE Multimate® multipurpose CLDS at several soaking time periods of 4, 6 and 8 hours. Following the soaking durations, the cysts were subcultured onto non-nutrient agar plates seeded with heat killed Escherichia coli and observed daily for 14 days for any evidence of excystation of the cyst to the trophozoite stage. Successful excystation indicates ineffectiveness of the CLDS and vice-versa. Result: OPTICARE Multimate® multipurpose CLDS with Polyhexamethylene Biguanide (PHMB) as active ingredient, failed to exhibit anti-Acanthamoeba activity in all soaking intervals against the 2 environmental isolates of Acanthamoeba. Conclusion: This study has proven that though contact lens disinfecting solutions have antimicrobial properties, not all are effective against Acanthamoeba cysts. OPTICARE Multimate® multipurpose CLDS may not be able to protect contact lens users from Acanthamoeba keratitis but more Acanthamoeba strains including the clinical, should further be tested. [ABSTRACT FROM AUTHOR]

• A. castellanii trophozoites are able to trigger NET formation upon in vitro interaction with neutrophils. • A. castellanii trophozoites degrade the NET DNA scaffold. • Trophozoites exhibit significantly higher 3′-NT/NU activity than cysts, which are not able to trigger NET release. Acanthamoeba castellanii is a free-living amoeba that acts as an opportunistic pathogen for humans and is the pathogenic agent of Acanthamoeba keratitis (AK). A. castellanii may present as proliferative and infective trophozoites or as resistant cysts during their life cycle. The immune response against AK is still poorly explored; however, it is well established that macrophages and neutrophils play essential roles in controlling corneal infection during the disease outcome. The release of NETs is one of the innate immune strategies to prevent parasite infection, especially when neutrophils interact with microorganisms that are too large to be phagocytosed, which is the case for amoeba species. The present work demonstrated that A. castellanii trophozoites can trigger NET formation upon in vitro interaction with neutrophils. Using DNase as a control, we observed increased parasite survival after coinciding with neutrophils, which may be correlated with NET degradation. Indeed, A. castellanii trophozoites degrade the NET DNA scaffold. Molecular analysis confirmed the occurrence of a 3′-nucleotidase/nuclease (3′-NT/NU) in the A. castellanii genome. We also demonstrated that trophozoites exhibit significantly higher 3′-NT/NU activity than cysts, which cannot trigger NET release. Considering that previous studies indicated the pathological role of 3′-NT-/NU in parasite infection, we suggest that this enzyme may act as the mechanism of escape of A. castellanii trophozoites from NETs. [ABSTRACT FROM AUTHOR]

• A. castellanii trophozoites have an acid ecto-phosphatase activity; • Sodium orthovanadate inhibits both the ecto-phosphatase activity and the adhesion of trophozoites to epithelial cells. • The incubation with mannose was able to stimulate ecto-phosphatase activity; Acanthamoeba castellanii is a free-living amoeba and an opportunistic pathogen for humans that can cause encephalitis and, more commonly, Acanthamoeba keratitis. During its life cycle, A. castellanii may present as proliferative and infective trophozoites or resistant cysts. The adhesion of trophozoites to host cells is a key first step in the pathogenesis of infection. A major virulence protein of Acanthamoeba is a mannose-binding protein (MBP) that mediates the adhesion of amoebae to cell surfaces. Ectophosphatases are ecto-enzymes that can dephosphorylate extracellular substrates and have already been described in several microorganisms. Regarding their physiological roles, there is consistent evidence that ectophosphatase activities play an important role in parasite-host interactions. In the present work, we identified and biochemically characterized the ectophosphatase activity of A. castellanii. The ectophosphatase activity is acidic, stimulated by magnesium, cobalt and nickel, and presents the following apparent kinetic parameters: K m = 2.12 ± 0.54 mM p -NPP and V max = 26.12 ± 2.53 nmol p -NP × h−1 × 10-6 cells. We observed that sodium orthovanadate, ammonium molybdate, sodium fluoride, and inorganic phosphate are able to inhibit ectophosphatase activity. Comparing the two stages of the A. castellanii lifecycle, ectophosphatase activity is significantly higher in trophozoites than in cysts. The ectophosphatase activity is stimulated by mannose residues and is significantly increased when trophozoites interact with LLC-MK2 cells. The inhibition of ectophosphatase by pretreatment with sodium orthovanadate also inhibits the adhesion of trophozoites to epithelial cells. These results allow us to conclude that the ectophosphatase activity of A. castellanii is somehow important for the adhesion of trophozoites to their host cells. According to our data, we believe that the activation of MBP by mannose residues triggers the stimulation of ectophosphatase activity to facilitate the adhesion process. [ABSTRACT FROM AUTHOR]

Introduction: Acanthamoeba keratitis (AK), caused by a pathogenic amoeba, is a sight-threatening corneal infection usually affecting contact lens wearers. Increasing numbers of Acanthamoeba keratitis cases are reported globally including Malaysia, along with increased use of soft contact lenses. However the true extent or exact frequency of the disease is not known in Malaysia since it is not a notifiable disease in this country. Objective: This study investigates the prevalence of Acanthamoeba keratitis from 2013-2015 at the Hospital Universiti Kebangsaan Malaysia (HUKM) in Kuala Lumpur, Malaysia. Materials and Methods: This retrospective study involved clinical samples submitted to the Acanthamoeba laboratory at the Department of Parasitology and Medical Entomology UKM from the year of 2013-2015. The study only included patients diagnosed as having microbial keratitis with Acanthamoeba as the presumptive causative agent. Records from the request forms for Acanthamoeba isolation and the culture results were analysed. Results: A total of 62 suspected cases had their corneal scrapings sent to the Acanthamoeba laboratory from different hospitals across Malaysia. 14 (22.6%) parasitologically confirmed culture positive cases were identified from the total 62 suspected cases during the period 2013-2015. All the 14 positive cases were contact lens wearers and they all presented with similar symptoms of redness, pain, watery eye and photophobia. All cases came from private hospitals throughout Malaysia. Majority of them (34.6%) were women and men constituted 13.8%. Conclusion: All 14 cases were diagnosed at only one study center and it would be anticipated that the number of cases would increase if there were more study centers involved throughout Malaysia. This study has proven that AK is not extremely rare in Malaysia and it should be considered as part of the differential diagnosis of most cases of presumed microbial keratitis especially those involving contact lens wearers. [ABSTRACT FROM AUTHOR]

Introduction: Acanthamoeba is a free-living amoeba found in abundance in all types of environments including soil, water and air. The risk for contracting Acanthamoeba keratitis (AK) is increased by exposure to environmental strains of Acanthamoeba through contact lens wear. Objective: The available contact lens disinfecting solutions in the market possess bactericidal and fungicidal properties but there is doubt about their amoebicidal properties which have inspired us to evaluate Renu® Sensitive, a new brand of multipurpose contact lens disinfecting solution (clds) in the market against Acanthamoeba cyst of environmental isolates. Materials and Methods: Suspension of cyst isolates from the environment was exposed to Renu® Sensitive multipurpose clds at intervals varying from 4 to 8 hours and later cultured onto non-nutrient agar lawned with Escherichia coli for the growth of Acanthamoeba trophozoites. Result: Renu® Sensitive multipurpose clds exhibited no cysticidal effect against cyst of Acanthamoeba spp from both environmental isolates in all tested soaking periods. Conclusion: This study had shown that the Acanthamoeba cysts of environmental isolates were resistant to Renu® Sensitive multipurpose clds (mclds) and the disinfectant might not protect the users from contracting Acanthamoeba keratitis [ABSTRACT FROM AUTHOR]

Introduction. Free-living amoebae, ubiquitous in outer environments, in predisposing circumstances may exist as parasites, infectious agents of Acanthamoeba keratitis. In recent decades, the vision-threatening corneal infection is a growing human health threat worldwide, including Poland. The applied therapy is often ineffective due to diagnostic mistakes, various pathogenicity of Acanthamoeba strains and high resistance of cysts to drugs; many agents with possible anti-amoebic activity are still being tested. In the presented study, selected chemicals are investigated in terms of their in vitro effect on corneal and environmental Acanthamoeba strains. Materials and method. Samples of a corneal isolate from a patient with severe Acanthamoeba keratitis, of assessed on the basis of genotype associations of 18S rRNA and the type strain, Acanthamoeba castellanii Neff cultivated in bacteria-free condition, were exposed to povidone iodine, chlorhexidine digluconate or toyocamycin. In vitro population dynamics of the strains were monitored and compared to those of control cultures. Results. All chemicals showed anti-amoebic effects with different degrees of effectiveness. Significant differences were observed in the in vitro population dynamics, and the morpho-physiological status of A. castellanii Neff T4 and corneal strains determined as A. polyphaga T4 genotype, exposed to povidone iodine or toyocamycin, in comparison with chlorhexidine taken as reference. Conclusions. Time-dependent amoebstatic in vitro effects were demonstrated for all agents, in particular, the results of assays with povidone iodine are promising. No significant stimulation of encystation appeared; however, as cysticidal efficacy of chemicals is expected, complementary research is needed on different Acanthamoeba strains with modified agent concentrations and method application. [ABSTRACT FROM AUTHOR]

Abstract A number of ocular diseases can be attributed to contaminated water and we have coined a term “Water-related ocular diseases (WRODs)” to denote this wide-spectrum of conditions. WRODs are directly related to human contact with water and can occur through toxic, allergic, inflammatory or infective mechanisms. The non-infective causes can include chemicals used to clean swimming pools, oil spills and water-sport related injuries. Similarly, a number of infective organisms causing ocular diseases are transmitted through water. Since, these conditions can occasionally prove devastating, a review was done with the following aims: (i) To study the epidemiology of WRODs (ii) To assess the clinical presentation and current management of WRODs (iii) To highlight the future challenges and possible solutions to these problems. The online search was conducted utilizing search engines such as PubMed, Google Scholar, ClinicalKey and the Virtual Library of the Ministry of Health, Malaysia for relevant terms such as water-borne, swimming pool and eye infections. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis (AK) is a severe corneal infection that may occur as a serious outcome of improper use of contact lenses (CL). Objectives The study aimed to diagnose AK in soft CL users presenting with infectious keratitis, and to identify the prevalent genotypes isolated from different cases. Another aim was to determine the CL hygiene-related risk behaviors, and to explore the risk of water exposure for developing AK. Methods A cross sectional study was performed. A questionnaire was carried out including 260 clinically diagnosed cases as infectious keratitis (170 females and 90 males); all of them were soft CL users for the suspected risk factors. Corneal scrapes from the affected eyes were cultured to diagnose bacterial and AK. PCR was performed and the amplified products were sequenced and compared with GenBank data. Results The parasite was positively amplified from 32 samples (12.3%). Acanthamoeba T4 genotype was identified in 27/32 (84.4%) of isolates. Other detected genotypes belonged to T5 and T3 genotypes at rates of 9.4%, and 6.25%, respectively. The most important risk factors associated with development of AK were female sex, sleeping while wearing CL, and exposure to water resources through different practices. These practices included rinsing the CL case in tap water, swimming and/or showering while wearing CL, using multipurpose solution for cleaning the lenses, using water from over-building tanks. Rubbing the eyes due to discomfort when applying CL was an additional important risk factor associated with AK. The protective factor was regular hand washing before using CL. Conclusion CL users were more exposed to AK and should gain enough health education regarding proper lens hygiene and dangers of tap water exposure. [ABSTRACT FROM AUTHOR]

In order to improve our knowledge on the epidemiology of amoebic keratitis, as well as the identification of Acanthamoeba isolates, we have isolated Acanthamoeba spp. from five symptomatic patients in Spain in the present study. All isolates were grown in axenic liquid medium, with only one exception. The morphology of these isolates were characterized by optical and scanning electron microscopy. Their structural features corresponded to those of amphizoic amoebae (namely Acanthamoeba spp.). The molecular characterization of the five Acanthamoeba isolates yielded six sequences. Almost complete 18S rRNA gene sequences (>2000 bp) were obtained from three isolates and partial sequences (∼1500 bp) from the other two. A robust phylogenetic analysis based on the almost complete 18S rRNA sequence showed that four isolates belonged to the T4 genotype and the other one to the T3 genotype. However, all isolates were identified as T4 genotype using the ASA.S1 fragment. As previously suggested by other researchers, only a robust phylogenetic approach may be helpful in identifying Acanthamoeba genotypes. In addition, new data on the phylogenetic relationships among the Acanthamoeba genotypes is provided and discussed. [ABSTRACT FROM AUTHOR]

Acanthamoeba species can cause a keratitis misdiagnosed as herpes keratitis or fungal keratitis. We report an unusual dendrite-like anterior stromal keratitis coinfected with Acanthamoeba and Pseudomonas aeruginosa in an orthokeratology contact lens wearer in Taiwan. Topical 1% voriconazole and 0.5% levofloxacin were prescribed because besides Acanthamoeba keratitis, fungal keratitis was also highly suspected initially. Topical 0.02% chlorhexidine was added after the culture of the scraped cornea showed positive results of Acanthamoeba and P. aeruginosa. The lesion subsided using this triple combination therapy for 1 week. Both Acanthamoeba and P. aeruginosa are potentially devastating causes of infectious keratitis. Our case highlights the importance of considering the possibility of a concurrent infection and atypical presentation in cases with contact lens-related keratitis. The use of topical levofloxacin combined with voriconazole should be considered as the first-line treatment in such patients. [ABSTRACT FROM AUTHOR]

Acanthamoeba castellanii is a free-living protozoan pathogen capable of causing a blinding keratitis and fatal granulomatous encephalitis. Current treatment generally involves an hourly application of polyhexamethylene biguanide (PHMB) over a period of several days but this is not entirely effective against all strains/isolates. The tolerance mechanisms of PHMB in Acanthamoeba cells remain unclear. In this study, we found that the mRNA expression level of disulfideisomerase domain containing protein (PDI) increased rapidly in surviving cells of the highly PHMB-tolerant Acanthamoeba castellanii strain, NCKH_D, during PHMB treatment, but not in the ATCC standard strain. After PDI-specific silencing, NCKH_D was found to be more vulnerable to PHMB treatment. The results described above show that PDI is an important gene for PHMB tolerance ability in a highly PHMB-tolerant strain of Acanthamoeba and provide a new insight for more efficient medicine development for Acanthamoeba keratitis. [ABSTRACT FROM AUTHOR]

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis. [ABSTRACT FROM AUTHOR]

Acanthamoeba, a free-living amoeba, is widely distributed in the environment, water sources, soil, dust, and air. It can cause keratitis in contact lens wearers with poor hygiene and also fatal granulomatous amebic encephalitis (GAE) in immunocompromised hosts. The aim of this study was to gain some insights into the distribution and genotypes of the potentially pathogenic species of Acanthamoeba present in water sources in north of Iran. Total 43 Acanthamoeba species were isolated from 77 water samples taken from different water sources within the Mazandaran province in Northern Iran (Sari city and suburbs). Isolates were identified based on cyst and trophozoite morphological characteristics as well genetics. PCR fragments corresponding to the small-subunit 18S rRNA gene were sequenced for 20 of 43 positive isolates. The results revealed that 83.3% of sequenced isolates belonged to the T4 genotype and the rest belonged to the T2 genotype. Our results indicated that Acanthamoeba is widely distributed in Sari city. As the incidence in Iran of amoebic keratitis has increased in recent years, the exact estimation of the prevalence of this amoeba and its predominant genotype may play a crucial role in prevention of the disease. Sari city has several rivers, seashores, and natural recreational amenities, which attract visitors during the year. This is the first report of Acanthamoeba genotypes from water sources in Sari city, Mazandaran province of Iran, and the results suggest that more attention is needed to protect the visiting population and immunocompromised individuals. [ABSTRACT FROM AUTHOR]

Background/purpose: Acanthamoeba keratitis is difficult to treat because Acanthamoeba cysts are resistant to the majority of antimicrobial agents. Despite the efficacy of 0.02% chlorhexidine in treating Acanthamoeba keratitis, a lack of data in the literature regarding the formulation's stability limits its clinical use. The objective of this study was to develop an optimal extemporaneous 0.02% chlorhexidine digluconate ophthalmic formulation for patients in need.Methods: With available active pharmaceutical ingredients, 0.02% chlorhexidine digluconate sample solutions were prepared by diluting with BSS Plus Solution or acetate buffer. Influences of the buffer, type of container, and temperature under daily-open condition were assessed based on the changes of pH values and chlorhexidine concentrations of the test samples weekly. To determine the beyond-use date, the optimal samples were stored at 2-8°C or room temperature, and analyzed at time 0 and at Week 1, Week 2, Week 3, Week 4, Week 5, Week 8, Week 12, and Week 24.Results: Despite chlorhexidine exhibiting better stability in acetate buffer than in BSS solution, its shelf-life was < 14 days when stored in a light-resistant low-density polyethylene container. The acetate-buffered 0.02% chlorhexidine digluconate solution stored in light-resistant high-density polyethylene eyedroppers did not exhibit significant changes in pH or strength at any time interval.Conclusion: The acetate-buffered 0.02% chlorhexidine digluconate ophthalmic solution stored in light-resistant high-density polyethylene eyedroppers demonstrated excellent stability at 2-25°C for 6 months after being sealed and for 1 month after opening. This finding will enable us to prepare 0.02% chlorhexidine digluconate ophthalmic solutions based on a doctor's prescription. [ABSTRACT FROM AUTHOR]

Acanthamoeba T4 genotype is the most prevalent genotype associated with amoebic keratitis. Acanthamoeba keratitis therapy is difficult due to transformation of trophozoite to cyst stage, which hinders the treatment of the disease. Although encystation assists the organism to survive against the chemotherapeutic compounds, the precise mechanism of encystation remains poorly understood. The purpose of this work was to identify differentially expressed genes in Acanthamoeba T4 genotype which might be useful for understanding of the encystment process and may thus help develop more efficient treatment. The mRNA profile of trophozoite and cyst of Acanthamoeba T4 genotype isolated from a soft contact lens wearer were analyzed using a cDNA amplified fragment length polymorphism (cDNA-AFLP) technique. Subsequently, a real time reverse transcriptase-PCR was performed to validate the cDNA-AFLP results. Three genes, heat shock protein70 ( hsp70) , actin-I and elongation factor-1alpha ( EF-1α ) were differentially expressed during Acanthamoeba differentiation. An in silico result predicted that transformation of trophozoite to cyst could be mediated through their cooperation with the protein partners interaction. Taken together, our experimental and bioinformatics findings suggested potential functions of hsp70, EF-1α and actin-I in differentiation of Acanthamoeba T4 genotype which may be useful in the design of an efficient therapeutic strategy in AK. [ABSTRACT FROM AUTHOR]