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Purpose The purpose of this article was to compare clinical outcomes between therapeutic penetrating keratoplasty (TPK), therapeutic deep anterior lamellar keratoplasty (TDALK), and optical penetrating keratoplasty (OPK) in Acanthamoeba keratitis. Methods A literature search was conducted in online libraries from 1980 to 2021. The primary end points were best-corrected visual acuity (VA), graft survival, and infection recurrence. In addition, we enrolled 35 consecutive patients with AK from our practice evaluating best-corrected VA and high-order aberrations. Results A total of 359 AK eyes from 33 published studies were retrieved from 175 publications screened. One hundred sixty-five eyes (73%) that underwent TPK and 39 eyes (84%) treated with TDALK had a clear graft at the last follow-up visit. Only the patients treated with OPK had 82 clear grafts (94%) during the follow-up period. Forty-seven (21%) of TPK patients reached VA ≥20/30, compared with 11 (25%) of TDALK patients and 35 (40%) of OPK patients. Acanthamoeba infection recurrence occurred in 38 eyes (16.8%) that underwent TPK, 9 (19%) that underwent TDALK, and 8 (9.5%) that underwent OPK. In our series, best-corrected visual acuity in nonsurgically treated patients was 1 ± 0.50 logMAR compared with 0 logMAR of surgically treated patients. High-order aberrations were significantly lower in surgically treated eyes after AK resolution, particularly in TDALK when compared with TPK patients. Best-corrected visual acuity was better in TDALK patients compared with TPK patients. Conclusions After AK resolution by 6 to 12 months of medical treatment, OPK seems to be the best surgical choice in patients with AK. If AK could not be eradicated by medical therapy, TDALK may be chosen in the early disease stage and TPK in later stages.

The aim of this study was to determine the impact of Acanthamoeba keratitis (AK) caused by contact lens (CL) use on vision-related quality of life (VRQOL) and the sociodemographic factors and disease outcome associated with VRQOL.Sixty-one CL-associated AK cases and 59 asymptomatic CL wearers (mean age ±SD 39.4 ± 16.5 vs. 45.5 ± 15.2 yrs, P = 0.04) were recruited from Moorfields Eye Hospital and Institute for Optometry, London. AK cases were surveyed during active disease and were stratified into "poor" and "good" outcomes based on clinical features. VRQOL was measured using Rasch-transformed scores from the Emotional, Mobility, and Reading domains of the 32-item Impact of Visual Impairment questionnaire. AK cases were compared with controls and "poor" outcomes compared with "good" with multivariable linear regression. Multivariable linear regression models were also used to identify the sociodemographic factors and disease outcome associated with VRQOL.AK was associated with significant and substantial reductions in all 3 evaluated domains of VRQOL (Reading -59.6%, Mobility -59.8%, and Emotional -66.2%) compared with controls, independent of sociodemographic factors. Patients with AK who experienced poor outcomes, those who were of British White race (compared with all other races) and female, had lower VRQOL scores across all domains. Patients with AK with lower incomes scored worse on Reading and Mobility domains, whereas those with lower education had poorer Emotional scores.AK has a considerable detrimental impact on VRQOL. Clinicians should consider the importance of referring patients with AK for rehabilitative support and counseling as part of active disease management.

PURPOSE The purpose of this study was to characterize rates of opioid prescription for different ulcerative keratitis types. METHODS This cohort study included patients diagnosed with ulcerative keratitis according to the University of Michigan electronic health record data between September 1, 2014 and December 22, 2020. Ulcerative keratitis was categorized by etiologic type (bacterial, fungal, viral, acanthamoeba, inflammatory, polymicrobial, or unspecified) using rule-based data classification that accounted for billing diagnosis code, antimicrobial or antiinflammatory medications prescribed, laboratory results, and manual chart review. Opioid prescriptions were converted to morphine milligram equivalent and summed over 90 days from diagnosis. Opioid prescription rate and amount were compared between ulcerative keratitis types. RESULTS Of 3322 patients with ulcerative keratitis, 173 (5.2%) were prescribed at least 1 opioid for pain management within 90 days of diagnosis. More patients with acanthamoeba (32.4%), fungal (21.1%), and polymicrobial (25.0%) keratitis were treated with opioids compared with bacterial (6.7%), unspecified (2.9%), or viral (1.8%) keratitis (all Bonferroni adjusted P < 0.05). For the 173 patients who were prescribed opioids, a total of 353 prescriptions were given within 90 days of diagnosis, with half given within the first week after diagnosis. The quantity of opioid prescribed within 90 days from diagnosis was not significantly different between ulcerative keratitis types (P = 0.6559). Morphine milligram equivalent units prescribed ranged from 97.5 for acanthamoeba keratitis to 112.5 for fungal keratitis. CONCLUSIONS The type of ulcerative keratitis may influence the opioid prescription rate. Providers can better serve patients needing opioids for pain management through improved characterization of pain and development of more tailored pain management regimens.

Context Acanthamoeba is increasingly implicated in causing keratitis in patients wearing contact lens or ocular trauma and has a poor prognosis. Establishment of an animal model is critical to study the disease pathology, pathogenesis and to evaluate anti-amoebic drugs. Some studies have used contact lenses to establish Acanthamoeba keratitis (AK) in a mouse model, which is expensive and not very successful as lenses get dislodged. Objective To assess the feasibility of using parafilm (Bemis Company Inc., USA) as an alternative to contact lens for the establishment of AK in the mouse model. Methods Thirty-six Balb/c mice in three groups of six mice each for two strains of Acanthamoeba were used to induce AK. Three experimental approaches used were; i) Acanthamoeba impregnated contact lens, ii) Acanthamoeba impregnated parafilm and iii) scratching followed by inoculation of Acanthamoeba suspension. In all three models, tarsorrhaphy was performed. Infection was evaluated by clinical examination and also through microscopic examination of corneal scrapings and corneal sections. Results AK model was successfully established with parafilm whereas only one mouse developed AK with the use of contact lens and none with scratching and Acanthamoeba inoculation. Conclusion The use of parafilm is convenient, reliable and cheaper and can be considered an alternative to contact lenses to induce AK in a mouse model.

Background Acanthamoeba keratitis (AK) is an important cause of ocular morbidity in both contact lens wearers and non wearers. Medical management comprises prolonged empiric treatment with multiple drugs, leading to adverse effects and suboptimal cure. The present study evaluated the efficiency and safety of common antimicrobial agents used in treatment of AK. Methods Six Acanthamoeba isolates (four AK, two water samples) were axenized and subjected to in vitro susceptibility testing against chlorhexidine, pentamidine isethionate, polymyxin B, miltefosine, and fluconazole to check for trophocidal and cysticidal activity. The safety profile was analysed by observing the cytotoxicity of the highest cidal concentration toward human corneal epithelial cell (HCEC) line. Results Chlorhexidine had the lowest cidal concentration against both cysts and trophozoites (range 4.16-25 μg/ml) followed by pentamidine isethionate (range 25-166.7 μg/ml). Both agents were nontoxic to HCEC. Polymyxin B (range 25-200 μg/ml) and fluconazole (range 64-512 μg/ml) had relatively higher minimum inhibitory concentrations (MIC); fluconazole was nontoxic even at 1024 μg/ml, but cytotoxicity was observed at 400 μg/ml with polymyxin B. Miltefosine was not effective against cysts at tested concentrations. A. castellanii were more susceptible to all agents (except pentamidine isethionate) than A. lenticulata. Clinical isolates were less susceptible to polymyxin B and fluconazole than environmental isolates, reverse was true for miltefosine. Conclusion Chlorhexidine and pentamidine isethionate were the most effective and safe agents against both trophozoites and cysts forms of our Acanthamoeba isolates. Fluconazole had higher MIC but was nontoxic. Polymyxin B was effective at high MIC but therapeutic dose was found toxic. Miltefosine, at tested concentrations, could not inhibit cysts of Acanthamoeba. Clinical isolates had higher MICs for polymyxin B and fluconazole.

Acanthamoeba spp. are among the most worldwide prevalent protozoa. It is the causative agent of a disease known as Acanthamoeba keratitis, a painful and severe sight-threatening corneal infection that can lead to blindness. In recent years, the prevalence of Acanthamoeba keratitis has rapidly increased, growing its importance to human health. This systematic review aims to assess the distribution of Acanthamoeba sp. genotypes causing keratitis around the world, considering the sample collected type and the used identification method. Most of the cases were found in Asia and Europe. Not surprisingly, the T4 genotype was the most prevalent worldwide, followed by T3, T15, T11, and T5. Furthermore, the T4 genotype contains a higher number of species. Given the differences in pathology, susceptibility to treatment, and clinical outcome between distinct genotypes, it is essential to genotype isolates from Acanthamoeba keratitis cases to help to establish a better correlation between in vitro and in vivo activities, resulting in better drug therapies and successful treatment in cases of this important ocular infection.

Efficacious treatments against Acanthamoeba Keratitis (AK) is challenging, often ineffective and linked to the intragenotype variation in the drug efficacy. Increased oxygen can facilitate host response and can inhibit some organisms. Herein, we report the effect of increased oxygen concentrations on Acanthamoeba spp. growth and its effect on ROS (reactive oxygen species) production. The exposition to pure oxygen could reduce cell growth by at least 60% for Acanthamoeba castellanii Neff, Acanthamoeba polyphaga, and Acanthamoeba griffini. The increase in ROS production confirming that oxygen cell’s growth inhibition was due to oxidative stress. Further studies are needed to determine oxygen saturation level, time of oxygen exposition, and number of sessions needed to eliminate the parasite.

Purpose: The aim of this study was to analyze patient data and the laboratory results of corneal samples collected from patients followed at the Ophthalmology Department, Hospital São Paulo, Brazil over a 30-year period, and correlate the analize with contact lens wearing. Methods: Corneal samples from patients diagnosed clinically with microbial keratitis between January 1987 and December 2016 were included in this study. Cultures that were positive for bacteria, fungi, and Acanthamoeba spp. were analyzed retrospectively. To ascertain if the number of patients with contact-lens-associated microbial keratitis (as a risk factor for microbial infection) changed over time, the analysis was divided into three decades: 1987-1996, 1997-2006, and 2007-2016. Information pertaining to patient gender and age, as well as type of organism isolated, were compared among these periods. Results: The corneal samples of 10.562 patients with a clinical diagnosis of microbial keratitis were included in the study, among which 1.848 cases were related to the use of contact lenses. The results revealed that the frequency of contact-lens-associated microbial keratitis increased over the last two decades. Overall, females had contact-lens-associated microbial keratitis more frequently (59.5%). Patients aged 19-40 years also had contact-lens-associated microbial keratitis more frequently in all study periods. Staphylococcus spp. and Pseudomonas spp. were the most frequent Gram-positive and Gram-negative bacteria, respectively, in the microbial keratitis groups. Among the fungal cases of microbial keratitis, filamentous fungi were the most frequent fungi over the entire study period, with Fusarium spp. being the most frequent fungi in the group with microbial keratitis not associated with contact lens wearing (p

Infectious keratitis (IK) is the 5th leading cause of blindness globally. Broad-spectrum topical antimicrobial treatment is the current mainstay of treatment for IK, though adjuvant treatment or surgeries are often required in refractory cases of IK. This systematic review aimed to examine the effectiveness and safety of adjuvant amniotic membrane transplantation (AMT) for treating IK. Electronic databases, including MEDLINE, EMBASE and Cochrane Central, were searched for relevant articles. All clinical studies, including randomized controlled trials (RCTs), non-randomized controlled studies and case series (n > 5), were included. Primary outcome measure was time to complete corneal healing and secondary outcome measures included corrected-distance-visual-acuity (CDVA), uncorrected-distance-visual-acuity (UDVA), corneal vascularization and adverse events. A total of twenty-eight studies (including four RCTs) with 861 eyes were included. When compared to standard antimicrobial treatment alone, adjuvant AMT resulted in shorter mean time to complete corneal healing (− 4.08 days; 95% CI − 6.27 to − 1.88; p p = 0.04) at 1 month follow-up in moderate-to-severe bacterial and fungal keratitis, with no significant difference in the risk of adverse events (risk ratio 0.80; 0.46–1.38; p = 0.42). One RCT demonstrated that adjuvant AMT resulted in better CDVA and less corneal vascularization at 6 months follow-up (both p

INTRODUCTION: Acanthamoeba encompasses several species of free-living ameba encountered commonly throughout the environment. Unfortunately, these species of ameba can cause opportunistic infections that result in Acanthamoeba keratitis, granulomatous amebic encephalitis, and occasionally systemic infection. AREAS COVERED: This review discusses relevant literature found through PubMed and Google scholar published as of January 2021. The review summarizes current common Acanthamoeba keratitis treatments, drug discovery methodologies available for screening potential anti-Acanthamoeba compounds, and the anti-Acanthamoeba activity of various azole antifungal agents. EXPERT OPINION: While several biguanide and diamidine antimicrobial agents are available to clinicians to effectively treat Acanthamoeba keratitis, no singular treatment can effectively treat every Acanthamoeba keratitis case. Efforts to identify new anti-Acanthamoeba agents include trophozoite cell viability assays, which are amenable to high-throughput screening. Cysticidal assays remain largely manual and would benefit from further automation development. Additionally, the existing literature on the effectiveness of various azole antifungal agents for treating Acanthamoeba keratitis is incomplete or contradictory, suggesting the need for a systematic review of all azoles against different pathogenic Acanthamoeba strains.

The incidence of Acanthamoeba keratitis has been increasing since the previous decades, especially among contact lens users. This infection is majorly caused by the use of ineffective contact lens disinfecting solution. Thus, this study was conducted to evaluate the in vitro effects of multi-purpose disinfecting solutions (MPDS) against Acanthamoeba trophozoites and cysts. Acanthamoeba genotype T4 isolated from contact lens paraphernalia and an environmental strains were propagated for trophozoite or cyst-containing culture and adjusted in final concentration of 1 x 105 cells/ml. Amoebicidal and cysticidal assays were conducted by incubating trophozoites and cysts with OPTI-FREE® Express®, ReNu® Fresh™, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive according to the manufacturer’s minimum recommended disinfectant time (MMRDT) for up to 12 h at 30 ⁰C. Trypan blue hemocytometer-based microscopic counts determined amoebicidal and cysticidal effects. The viability of Acanthamoeba trophozoites and cysts was confirmed by re-inoculated them in the 1.5% non-nutrient agar plates. It was found that none of the MPDS showed amoebicidal and cysticidal effects during the MMRDT. However, OPTI-FREE® Express® demonstrated a significant differences in average cell reduction for both stages within MMRDT. When subjected to 12 h exposure, both OPTI-FREE® Express® and ReNu® Fresh™ led to significant reduction in the number of trophozoite and cyst cells. Notably, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive did appreciably improve the solution effectiveness towards trophozoite cells when incubated for 12 h. All MPDS were largely ineffective, with 100% survival of all isolates at MMRDT, while OPTI-FREE® Express® showed limited amoebicidal activity against the contact lens paraphernalia isolate, however, it was more against the environmental strains after 12 h incubation time. The commercially available MPDS employed in this research offered minimal effectiveness against the protozoa despite the contact time. Improvement or development of new solution should consider the adjustment of the appropriate disinfectant concentration, adequate exposure time or the incorporation of novel chemical elements, which are effective against Acanthamoeba for accelerated disinfecting and more reduction of potential exposure of contact lens users to Acanthamoeba keratitis.

The main corneal infections reported worldwide are caused by bacteria and viruses but, recently, the number of Acanthamoeba keratitis (AK) cases has increased. Acanthamoeba genus is an opportunistic free living protozoa widely distributed in environmental and clinical sources, with two life-cycle stages: the trophozoite and the cyst. AK presents as primary symptoms eye redness, epithelial defects, photophobia and intense pain. An early diagnosis and an effective treatment are crucial to avoid blindness or eye removal but, so far, there is no established treatment to this corneal infection. Diverse research studies have reported the efficacy of commercialized eye drops and ophthalmic solutions against the two life cycle stages of Acanthamoeba strains, that usually present preservatives such as Propylene Glycol of Benzalkonium chloride (BAK). These compounds present toxic effects in corneal cells, favouring the inflammatory response in the so sensitive eye tissue. In the present work we have evaluated the efficacy of nine proprietary ophthalmic solutions with and without preservatives (ASDA Dry Eyes Eyedrops, Miren®, ODM5®, Ectodol®, Systane® Complete, Ocudox®, Matrix Ocular®, Alins® and Coqun®) against the two life cycle stages of three Acanthamoeba strains. Our work has demonstrated the high anti-Acanthamoeba activity of Matrix Ocular®, which induces the programmed cell death mechanisms in Acanthamoeba spp. trophozoites. The high efficacy and the absence of ocular toxic effects of Matrix Ocular®, evidences the use of the Arabinogalactan derivatives as a new source of anti-AK compounds., Graphical abstract Image 1

Acanthamoeba keratitis (AK) is a rare but serious infection of the eye and can lead to blindness. The effective and safe medical therapy remains unclear for AK until present. Antimicrobial activity and biological characteristic of chitosan encourage screening of it against Acanthamoeba. Thus, in vitro anti-amoebic activities of commercial chitosan and nano-chitosan were tested on pathogenic Acanthamoeba genotype T4, a causative agent of human AK. The Acanthamoeba spp. was isolated from the keratitis patient. The Acanthamoeba genotype T4 was approved using PCR method followed by sequencing technique. Chitosan nanoparticles was prepared using ionic gelation method and characterized by their physicochemical properties. In the present study, the in vitro activity of serial dilutions (12.5, 25, 50, 100, and 200 µL/mL) of commercial chitosan and nano-chitosan were evaluated against Acanthamoeba trophozoites and cysts. The finding of nano-chitosan particle size by DLS was 118 nm with a PDI of about 0.134. Zeta potential value was found to be 42.7 mV. The obtained results showed that the tested chitosan and nano-chitosan presented anti-amoebic activities dependent to time and concentration. The inhibitory effect of the chitosan and nano-chitosan is enhanced by increasing the concentration and incubation time. The inhibitory effect of nano-chitosan on both trophozoites and cyst was more than chitosan. According to the results, nano-chitosan shows the potent activity against Acanthamoeba T4 and could be used for the development of novel and safe therapeutic approaches in the future.

Acanthamoeba keratitis is a sight-endangering eye infection, and causative organism Acanthamoeba presents a significant concern to public health, given escalation of contact lens wearers. Contemporary therapy is burdensome, necessitating prompt diagnosis and aggressive treatment. None of the contact lens disinfectants (local and international) can eradicate Acanthamoeba effectively. Using a range of compounds targeting cellulose, ion channels, and biochemical pathways, we employed bioassay-guided testing to determine their anti-amoebic effects. The results indicated that acarbose, indaziflam, terbuthylazine, glimepiride, inositol, vildagliptin and repaglinide showed anti-amoebic effects. Compounds showed minimal toxicity on human cells. Therefore, effects of the evaluated compounds after conjugation with nanoparticles should certainly be the subject of future studies and will likely lead to promising leads for potential applications.

This article presents an overview of information regarding Acanthamoeba keratitis per epidemiology, host-pathogen interactions, clinical manifestations, risk factors, environmental implications, diagnosis, treatment and management as well as COVID-19 characteristics which may be taken into cognizance for suspected infected patients, researchers and ophthalmologic practitioners. Acanthamoeba spp. is pathogenetically ubiquitous in nature. Acanthamoeba keratitis is considerably an ocular-threatening and debilitating keratitis that exhibits contumacious characteristics which hinder or impede treatment or management. At inception, Acanthamoeba generally depicts atypical clinical features which are frequently misdiagnosed as other microbial keratitis. Fundamentally, it constitutes a rare corneal infection of which the aetiologic agent is the protozoon Acanthamoeba spp. in contact lens wearers, presenting features of severe ophthalmic distress, blurred vision, blepharospasm, ocular excoriation, extraneous entity sensation and photophobia culminating in aberrant visual functionality. These are perspicuously due to retarded prompt and adequate treatment and management. Personal and environmental hygiene, especially on the hands, face and ocular areas as advised for the COVID-19 protocol could prevent contamination and dissemination of Acanthamoeba keratitis infection. The differentiating relatedness of Acanthamoeba keratitis, COVID-19 ophthalmologic infections and other ocular problems may not have been clearly elucidated.

Purpose To report a case series of patients with treatment-resistant Acanthamoeba keratitis (AK) using oral miltefosine, often as salvage therapy. Design Descriptive retrospective multi-center case series. Methods We reviewed 15 patients with AK unresponsive to therapy that were subsequently given adjuvant systemic miltefosine between 2011 to 2017. The main outcome measures were resolution of infection, final visual acuity, tolerance of miltefosine, and clinical course of disease. Results All patients were treated with biguanides and/or diamidines or azoles without resolution of disease prior to starting miltefosine. 11 of 15 patients retained count fingers or better vision, and all were considered disease-free at last follow up. 11 of 15 patients had worsening inflammation with miltefosine, with 10 of them improving with steroids. Six patients received multiple courses of miltefosine. Most tolerated oral miltefosine well, with mild gastrointestinal symptoms as the most common systemic side effect. Conclusion Oral miltefosine is a generally well-tolerated treatment adjuvant in patients with refractory acanthamoeba keratitis. The clinician should be prepared for a steroid-responsive inflammatory response frequently encountered during the treatment course.

Background: The genus Acanthamoeba is a free-living opportunistic protozoan parasite, which widely distributed in soil and fresh water. Acanthamoeba keratitis, which causes a sight-threating infection of the cornea, is going to rise in Iran and worldwide. The aim of this study was to compare direct microscopy, culture and PCR for detection of Acanthamoeba spp. in clinical samples and to determine the genotypes of Acanthamoeba spp. by sequencing 18SrRNA gene. Methods: Among patients clinically suspected to AK referred to a tertiary ophthalmology center at Mashhad, northeastern Iran. During 2017-18, twenty corneal scrapes specimens obtained. The samples were divided into three parts, subjected to direct microscopic examination, culture onto non-nutrient agar and PCR technique. Sensitivity, specificity, accuracy and likelihood ratio were evaluated. Results: Among 20 persons clinically suspected to amoebic keratitis, 13(69.2%) patients definitely diagnosed as Acanthamoeba keratitis. Wearing contact lens, eye trauma due to foreign particle and swimming in fresh water were the main predisposing factors. Most of patients suffered from pain and photophobia. Corneal ring infiltration and epithelial defect were common clinical sings. Direct examination had the lowest sensitivity and sensitivity of both Nelson-PCR and JDP-PCR methods were equal and highest. In addition, the results of sequencing identified that all strains belonged to T4 genotype. Conclusion: Amoebic keratitis is a sporadic parasitic keratitis, which is mainly seen in contact lens user in Mashhad. PCR based on 18S ribosomal DNA with JDP primers is a reliable and highly sensitive method for diagnosis of Acanthamoeba keratitis in clinically suspected cases.

Koji Hirano,1 Hidenori Tanaka,2 Kumiko Kato,3 Kaoru Araki-Sasaki4 1Department of Ophthalmology, Fujita Health University Bantane Hospital, Nagoya, Aichi, 454-8509, Japan; 2Department of Ophthalmology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192, Japan; 3Department of Ophthalmology, Mie University Graduate School of Medicine, Tsu, Mie, 514-8507, Japan; 4Department of Ophthalmology, Kansai Medical University, Hirakata, Osaka, 573-1010, JapanCorrespondence: Koji HiranoDepartment of Ophthalmology, Fujita Health University Bantane Hospital, 6-10, Otoubashi 3-cho-me, Nakagawa-ku, Nagoya, Aichi, 454-8509, JapanTel +81-52-321-8171Fax +81-52-322-4734Email kojihira@fujita-hu.ac.jpPurpose: In clinical practice we sometimes encounter patients with severe corneal ulcers who have been treated with topical corticosteroids. This study reviewed the clinical features and visual outcomes of these patients and investigated the background of the prescription of topical corticosteroids.Patients and Methods: The medical records of patients who visited the Cornea Service at Fujita Health University Bantane Hospital and were treated for infectious keratitis from April 2016 to March 2020 were retrospectively reviewed. Patients treated with topical corticosteroids before a culture-proven diagnosis were studied in terms of demographics, best-corrected visual acuity at arrival and at last visit, the clinical course after visit, ocular history, and combination therapy by the previous ophthalmologist.Results: Out of the 200 eyes of 197 patients with infectious keratitis, 14 eyes of 14 patients were treated with topical corticosteroids before a culture-proven diagnosis. All 14 patients were referred, as they had severe keratitis that could not be cured with topical antibiotics and corticosteroids. Based on the culture results, we diagnosed Acanthamoeba keratitis (AK) in six patients, fungal keratitis (FK) in two patients, bacterial keratitis (including a suspected case) in two patients, and unknown cause in four patients. Two patients with AK, FK, and unknown keratitis had unfortunate clinical courses and poor visual outcomes. From the information in the referral letters, at least six of the 14 patients were treated with either acyclovir ocular ointment or valaciclovir tablets, along with topical corticosteroids.Conclusion: Application of topical corticosteroids for keratitis that does not respond to empirical antibiotic therapy is harmful since AK or FK is likely involved in these topical antibiotic-resistant cases. Microbiological evidence, as well as a differential diagnosis of herpetic stromal keratitis, is needed when prescribing topical corticosteroid for the treatment of suspected infectious keratitis.Keywords: culture-proven diagnosis, empiric antibiotic therapy, herpetic keratitis, infectious keratitis, topical corticosteroids

Acanthamoeba keratitis with bacterial, fungal superinfection or without it leads to development of an aggressive and long-standing corneal inflammation; up to now, the efficacy of its treatment stays doubtful and demands further investigation. For a long time, there were discussions on the possibility and expediency of corneal collagen cross-linking (PACK-CXL photo activated chromophore for keratitis) in patients with bacterial, fungal and Acanthamoeba keratitis. This article presents a clinical case of effective treatment of mixed etiology keratitis by multiple high fluence accelerated PACK-CXL in a patient with severe local toxico-allergic reaction.

Corneal opacity is the 5th leading cause of blindness and visual impairment globally, affecting ~6 million of the world population. In addition, it is responsible for 1.5-2.0 million new cases of monocular blindness per year, highlighting an ongoing uncurbed burden on human health. Among all aetiologies such as infection, trauma, inflammation, degeneration and nutritional deficiency, infectious keratitis (IK) represents the leading cause of corneal blindness in both developed and developing countries, with an estimated incidence ranging from 2.5 to 799 per 100,000 population-year. IK can be caused by a wide range of microorganisms, including bacteria, fungi, virus, parasites and polymicrobial infection. Subject to the geographical and temporal variations, bacteria and fungi have been shown to be the most common causative microorganisms for corneal infection. Although viral and Acanthamoeba keratitis are less common, they represent important causes for corneal blindness in the developed countries. Contact lens wear, trauma, ocular surface diseases, lid diseases, and post-ocular surgery have been shown to be the major risk factors for IK. Broad-spectrum topical antimicrobial treatment is the current mainstay of treatment for IK, though its effectiveness is being challenged by the emergence of antimicrobial resistance, including multidrug resistance, in some parts of the world. In this review, we aim to provide an updated review on IK, encompassing the epidemiology, causative microorganisms, major risk factors and the impact of antimicrobial resistance.摘要: 角膜混浊是全球致盲和视力障碍的第五大原因, 世界范围内大约600万人受其影响。此外, 角膜混浊每年导致的单眼失明约150-200万例, 突显其对人类健康造成的持久性负担。在感染、创伤、炎症、变性和营养缺乏等所有的致病因素中, 感染性角膜炎 (IK) 是发达国家和发展中国家角膜病致盲的主要原因, 大概每100000人口中2.5-799人罹患此病。IK可由多种微生物引起, 包括细菌、真菌、病毒、寄生虫和多重感染。受地理和时间变化的影响, 细菌和真菌已被证明是角膜感染最常见的病原微生物。虽然病毒性角膜炎和棘阿米巴角膜炎并不常见, 但在发达国家, 它们是角膜病致盲的重要原因。接触镜的佩戴、外伤、眼表疾病、眼睑疾病以及眼部手术已证实是IK的主要危险因素。广谱抗生素是目前IK治疗的主要选择, 但是在世界某些地区, 其有效性正在受到抗菌药物耐药性的挑战, 其中包含多重耐药性等。本篇综述旨在提供有关IK的最新进展, 包括流行病学、病原微生物、主要危险因素以及抗生素耐药性对于疗效的影响。.

Acanthamoeba keratitis is a serious infection of the eye that can result in permanent visual impairment or blindness, caused by free-living amoebae of the genus Acanthamoeba. Early diagnosis is necessary for effective treatment of Acanthamoeba keratitis. Acanthamoeba is abundant in nature and can be found in water, soil, and air. Acanthamoeba keratitis is usually diagnosed by culture from a scraping of the eye or by confocal microscopy. In this paper, two complicated Acanthamoeba keratitis cases are reported.

Introduction: Acanthamoeba keratitis is a serious infection of the cornea which causes severe inflammation of the eye and loss of vision. Case description: A 45 years female came with a history of trauma of right eye with vegetative matter with severe pain and redness. Patient was treated with antibacterial, antifungal eye drops but the condition deteriorated. Diagnosis was based on presence of motile trophozoites and cyst in the culture of non nutrient agar with a lawn of E coli of the corneal ulcer scraping. Therapeutic penetrating keratoplasty was performed as the patient’s condition deteriorated. Conclusion: Acanthamoeba keratitis is a rare parasitic vision threatening infection with poor prognosis in comparison to other infectious keratitis. Therefore prevention is very important.

Objectives To report the negative fluorescein staining as an early sign of Acanthamoeba keratitis (AK). Methods Case report and brief review of related literature. Report of case A 30-year-old, contact lens wearer, woman presented with mild irritation and pain in the right eye. The best-corrected visual acuity (BCVA) was 20/20 in both eyes. Slit-lamp examination revealed a peripheral corneal infiltration. Empirical antimicrobial therapy was initiated. Within the third day, peripheral corneal opacity regressed but a Y-shaped linear epitheliopathy with a negative fluorescein staining, because of a ridge-like epithelial irregularity, was observed in the central cornea. Clinical findings progressed rapidly. Confocal microscopy revealed hyper-reflective cysts with the typical double-ring sign consistent with AK. Therefore, topical chlorhexidine and propamidine were initiated. Clinical findings regressed subsequently. The final BCVA was 20/20 in both eyes. Conclusion Acanthamoeba keratitis usually manifest as superficial epitheliopathy and progresses to the stroma. Findings may be obscure or atypical; comprehensive and careful examination may reveal mild findings in the early stages.

Introduction: Acanthamoeba keratitis (AK), a potentially devastating disease is caused by free-living amoebae of the genus Acanthamoeba . The number of reported cases worldwide is increasing year after year, mostly in contact lens wearers as also in non-contact lens wearers. Interestingly, AK has remained significant due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. Corneal blindness is a major public health problem worldwide and infectious keratitis is one of the predominant causes. Aim: To find out the Parasitic Etiological Agents in Suppurative Keratitis at a tertiary eye care referral centre in South India. Materials and Methods: A hospital based study was done on total of 350 cases of keratitis, during the period from January 2013 to December 2013 .The samples were examined as wet mount as well as cultured onto Non nutrient agar with E.coli overlay. Result: Acanthamoeba species were isolated from 314 cases. Most of the corneal ulcers responded well to treatment which was started early in their course due to early detection of the parasite by direct microscopy and confirmation by culture. Keywords: Acanthamoeba spp, Etiological Agents, Suppurative keratitis.

Background and objectives Acanthamoeba keratitis is a sight-threatening infectious disease that is difficult to treat. The aim of this study was to evaluate TONS504 (cationic chlorin derivative photosensitizer)-mediated photodynamic antimicrobial chemotherapy (PACT) in vivo as a potential treatment for Acanthamoeba keratitis. Study design/materials and methods Acanthamoeba keratitis was induced by soft contact lenses incubated with 1 × 105 /ml Acanthamoeba castellanii, which were placed over debrided corneas with temporary tarsorrhaphy. Thirty-eight male Japanese white rabbits were randomly divided into three groups (normal eye, no treatment, and treatment groups). TONS504 was administered as eye drops at 1 mg/ml, followed by light-emitting diode irradiation after the establishment of keratitis at 7 days after infectious contact lens exposure. All animals were evaluated under a slit-lamp microscope every 3 days for 6 days after the treatment. Clinical scores based on corneal epithelial defects detected by fluorescein staining, stromal opacity edema, and vascular infiltration into the cornea were determined. After 6 days, the eyes were enucleated for histopathological analysis. Results Clinical signs of infection in the treatment group were markedly reduced for up to 6 days after treatment. Histopathology showed a regular arrangement of stromal fibers and a small number of inflammatory cells in 58% of the corneas. However, 42% of corneas in the treatment group showed infiltrating neutrophils and irregular alignment of stromal collagen fibers. Conclusions Our TONS504-PACT achieved complete recovery from keratitis in 58% of the rabbit models. Further studies are required to determine the conditions for the maximal effectiveness of our TONS504-PACT for Acanthamoeba keratitis. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

A 28-year-old woman with a history of trauma to her right eye 2 months prior reported experiencing a stinging sensation and tearing in the morning since the injury occurred and the need to occasionally use an eye patch. Three days before presentation she had been prescribed a therapeutic contact lens (CL) with the diagnosis of a corneal epithelial defect. She described significant pain despite the CL. There was a corneal lesion with haze at the base surrounded by corneal edema. Corneal confocal images revealed hyperreflective cystic lesions that suggested Acanthamoeba keratitis (AK). However, the lesion healed within 10 days and the results of cultures taken before the initiation of treatment proved to be negative. The history of trauma and CL wear, the presence of severe pain, corneal findings, and the confocal microscopy detection of cysts led to a suspicion of AK in a differential diagnosis, but the final diagnosis was recurrent epithelial erosion based on the negative culture results, quick response to treatment, and the possibility of similar confocal findings in a healing epithelium. Since AK may cause loss of vision, suspicion should require that samples be obtained for microbiological study and close follow-up of the clinical course until a final diagnosis can be achieved.

Interest in periocular (eyelid and eyelashes margins) hygiene has attracted attention recently and a growing number of commercials eye cleanser and shampoos have been marketed. In the present study, a particular eye cleanser foam, Belcils® has been tested against trophozoites and cysts on the facultative pathogen Acanthamoeba. Viability was tested by the alamarBlue™ method and the foam was tested for the induction of programmed cell death in order to explore its mode of action. We found that a 1% solution of the foam eliminated both trophozoite and cyst stage of Acanthamoeba spp. After 90 min of incubation, Belcils® induced, DNA condensation, collapse in the mitochondrial membrane potential and reduction of the ATP level production in Acanthamoeba. We conclude that the foam destroys the cells by the induction of an apoptosis-like process. The current eye cleanser could be used as part of AK therapy protocol and as prevention from AK infections for contact lens users and post-ocular trauma patients., Graphical abstract Image 1

Background and AimsPolyhexamethyl biguanide (PHMB), a widely used topical treatment for Acanthamoeba keratitis (AK), is unlicensed with no formal safety assessment. This study evaluated its safety and tolerability.MethodsA prospective, randomised, double-masked controlled trial in 90 healthy volunteers. Subjects were treated with topical 0.04%, 0.06%, 0.08% PHMB or placebo (vehicle) 12× daily for 7 days, then 6× daily for 7 days. The rates of dose-limiting adverse events (DLAEs) leading to interruption of dosing, mild adverse events (AEs) (not dose limiting) and incidental AEs (unrelated to treatment) were compared. The primary outcome was the difference between treatments for DLAE rates.Results5/90 subjects developed DLAE within ConclusionThese findings are reassuring for PHMB 0.02% users. They also suggest that higher PHMB concentrations may show acceptable levels of tolerance and toxicity in AK subjects, whose susceptibility to AE may be greater than for the normal eyes in this study. Given the potential benefits of higher PHMB concentrations for treating deep stromal invasion in AK, we think that the use of PHMB 0.08% is justified in treatment trials.Trial registration numberNCT02506257.

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

This study evaluated in vitro effect of different concentrations of Aloe vera (A. vera) ethanol extract and honey against Acanthamoeba spp. cysts in comparison with chlorhexidine (the drug of choice for treatment of Acanthamoeba infection) at different incubation periods. Four different concentrations of the tested agents were used, 100, 200, 400, and 600 μg/ml for A. vera ethanol extract and 25, 50, 100, and 200 μg/ml for honey. Isolated Acanthamoeba spp. cysts from keratitis patients were incubated with different concentrations of the tested agents as well as chlorhexidine 0.02% (drug control) for different incubation periods (24, 48, 72 h). After each incubation period, the effect of A. vera extract and honey against Acanthamoeba cysts was assessed by counting the number of viable cysts, determining the inhibitory percentage and detecting the morphological alternations of treated cysts compared to non-treated and drug controls. Both A. vera ethanol extract and honey showed a concentration and time-dependent effect on the viability of Acanthamoeba cysts. In comparison with chlorhexidine (the drug control), A. vera ethanol extract possessed a potent cysticidal activity at all tested concentrations throughout different incubation periods, except for concentration 100 μg/ml which recorded the lower inhibitory effect. With increasing the dose of A. vera ethanol extract to 200, 400, 600 µg/ml, the recorded inhibitory percentages of Acanthamoeba cysts viability were 82.3%, 92.9% and 97.9% respectively, after 72 h compared to 76.3% of chlorhexidine. Similarly, honey at concentrations of 50–100 µg/ml gave higher inhibitory effect of 59% and 76.7%, respectively compared to chlorhexidine which showed an inhibitory percentage of 55.7% after 24 h. Meanwhile, the lowest tested concentration of honey (25 µg/ml) gave an inhibitory effect by 47.7–67% which was less than that of chlorhexidine throughout different incubation periods. With increasing the dose of honey to 200 µg/ml, the inhibitory effect was 98.9% after 72 h higher than that of chlorhexidine (76.9%). Using a scanning electron microscope, Acanthamoeba cysts treated by A. vera ethanol extract showed alternations in their shapes with flattening, collapsing, and laceration of their walls. Also, treated cysts by honey were highly distorted and difficult to identify because most of them were shrinkage and collapsed to a tiny size. On the other hand, chlorhexidine showed less structural and morphological changes of Acanthamoeba cysts. A. vera ethanol extract and honey had considerable cysticidal effects on Acanthamoeba cysts. They may give promising results for treatment of Acanthamoeba keratitis.

AimTo describe the characteristic clinical features and management of keratitis in the patients receiving miltefosine for post-kala-azar dermal leishmaniasis (PKDL).MethodsThe medical records of five patients with PKDL who presented with keratitis were reviewed retrospectively from April 2018 to December 2019. The evaluation included a thorough medical history including details on drugs used, particularly miltefosine. The drug causality assessment was also performed. The clinical and microbiological characteristics of keratitis were noted.ResultsThe ocular symptoms included pain, redness, watering, photophobia and diminution of vision. Slit-lamp biomicroscopy revealed peripheral, paralimbal, ring-shaped, full-thickness stromal infiltration resulting in ulcerative keratitis in all cases. Two patients had unilateral keratitis, while three had bilateral keratitis. All five patients received miltefosine for an average period of 48 days before the onset of keratitis. The corrected distance visual acuity at presentation ranged from hand movement to 20/125. The causality assessment revealed a ‘probable’ association between the adverse drug reaction and miltefosine in all patients. Discontinuation of miltefosine and initiation of corticosteroid therapy resulted in resolution of keratitis in all cases. The unilateral keratitis treated with topical corticosteroids had improved outcomes, but poor outcomes were found in the bilateral keratitis.ConclusionThese observations indicate that prolonged use of miltefosine might cause keratitis that resembles infectious keratitis. Early diagnosis with discontinuation of the drug and initiation of corticosteroid therapy are the key to successful management.

PURPOSE To report endotheliitis as an early, key presenting sign of Acanthamoeba keratitis in patients who are soft contact lens wearers. METHODS We report 4 cases of patients presenting with pain, red eye, and reduction in vision in the context of soft contact lens wear. On examination, the predominant clinical finding was that of endotheliitis, localized fusiform stromal edema with corresponding keratic precipitates, anterior chamber inflammation, and minimal epithelial and anterior stromal signs. The classical signs of Acanthamoeba keratitis were not present. RESULTS All 4 cases were clinically diagnosed as Acanthamoeba endotheliitis; corneal scrapes were negative; case 1 was polymerase chain reaction positive, and case 3 underwent confocal microscopy that showed double-walled cysts, suggesting Acanthamoeba. All responded well to anti-Acanthamoeba medication alone with 3 cases achieving complete resolution with minimal anterior stromal scarring by 7 weeks. Case 1 had steroid treatment before being seen at our unit and had a prolonged course of treatment with complete resolution by 4 months. CONCLUSIONS It is imperative to have a high index of suspicion for Acanthamoeba in patients presenting with pain and endotheliitis in the context of contact lens wear, even in the absence of classical signs.

Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta-oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully.

A 35-year old soft contact lens wearer with a proven bilateral Acanthamoeba keratitis developed a nodular scleritis. Based on the stepladder approach described by Iovieno et al. [Ophthalmology. 2014 Dec;121(12):2340–7], nonsteroidal anti-inflammatory drugs, methylprednisolone, and later azathioprine were added to the antiamoebic treatment. Unfortunately, there was further deterioration and an endophthalmitis developed. Unbearable pain and concerns of spread to the brain urged an enucleation. Histopathological examination confirmed Acanthamoeba cysts in the cornea, sclera, retina, choroid, and vitreous body. As a side effect of the immunosuppressive treatment, the patient developed myopathy, pulmonary aspergillosis, and an avascular necrosis of the hip. Scleritis is a devastating complication of Acanthamoeba keratitis with a poor prognosis and a high enucleation rate. Acanthamoeba sclerokeratitis is, due to cyst-free biopsies, mostly assigned to an immune-mediated mechanism, justifying the use of immunosuppressive treatment. Scleritis in our case contributed to the extracorneal spread of Acanthamoeba. Our case is the first documented extracorneal spread of Acanthamoeba without previous surgery. Extracorneal spread of Acanthamoeba should be considered, even in the case of false-negative biopsies. We strongly recommend serial sections of the retrieved scleral specimen in case of negative histopathological examination to exclude an infection. Even when an immune-mediated scleritis is suspected, systemic immunosuppressive treatment should always be used with the greatest caution. Awareness of the side effects and monitoring by an experienced physician is mandatory.

With less than 3 new cases per million people, Acanthamoeba keratitis (AK) is an orphan disease. It is a potentially devastating ocular infection without standardized guidelines for diagnostics and...

Acanthamoeba can cause visually destructive Acanthamoeba keratitis (AK) in contact lens (CL) users. The purpose of this study was to determine whether Acanthamoeba was present in the CL cases of CL wearers and to develop techniques to prevent the contaminations. To accomplish this, 512 CL case samples were collected from 305 healthy CL wearers. Using real-time PCR, Acanthamoeba DNA was detected in 19.1% of CL cases, however their presence was not directly associated with poor CL case care. Instead, the presence of Acanthamoeba DNA was associated with significant levels of many different bacterial species. When the CL cases underwent metagenomic analysis, the most abundant bacterial orders were Enterobacteriales followed by Burkholderiales, Pseudomonadales, and Flavobacteriales. The presence of Acanthamoeba was characterized by Propionibacterium acnes and Rothia aeria and was also associated with an increase in the α diversity. Collectively, Acanthamoeba contamination occurs when a diversified bacterial flora is present in CL cases. This can effectively be prevented by careful and thorough CL case care.

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

To report our experience with oral miltefosine (MLT) as an adjunct treatment for progressive Acanthamoeba keratitis (AK).Retrospective case series of all patients who underwent treatment with oral ...

Purpose To compare the clinical profiles and outcomes of patients with Acanthamoeba keratitis with or without mixed bacterial infection. Methods A retrospective analysis of microbiologically confirmed AK cases presenting to a tertiary care center over a 9-year period was conducted. Fourteen eyes in the coinfected group (group 1) and 24 eyes in the AK group (group 2) were analyzed to study the differences in these 2 groups of patients. The cases were diagnosed using a conventional microscopic analysis with staining techniques confirmed by cultural methods and were treated, which tailored to the microbiology report. Results There was no difference in the demographic profile and presenting features in the 2 groups. Duration of symptoms and history of ocular trauma, contact lens wear, and previous steroid usage were also similar for both the groups. Dense central corneal infiltrate was the common presentation for both groups (7/14 vs. 16/24 in group 1 vs. group 2). Epithelial defect without dense stromal infiltrate was significantly more common in group 1 (42.9% vs. 4.2%, P = 0.003) as a presenting clinical feature. Hypopyon was present in 8/14 of group 1 versus 9/24 of group 2 eyes (P = 0.25). No significant difference was found in the final visual acuity in pure and mixed AK cases, and the need for surgical intervention was comparable. The most common bacterial pathogen isolated in the mixed group was the Staphylococcus species. Conclusions Bacterial coinfection is common in patients with Acanthamoeba keratitis. Coinfection did not point toward a worse clinical disease at presentation or outcome.

ZusammenfassungDie Akanthamöbenkeratitis ist ein „Chamäleon“. Sie präsentiert sich klassischerweise mit gräulichen Epithelveränderungen, Perineuritis, oberflächlichen multifokalen stromalen Infiltraten und Ringinfiltrat. Bei klinischem Verdacht auf Akanthamöbenkeratitis ist eine Polymerase-Ketten-Reaktion, eine Kultivierung oder die histopathologische Untersuchung zur Diagnosestellung notwendig. Die konfokale Mikroskopie hat sich als noninvasive Ad-hoc-Diagnostik bei Initialverdacht instituiert.

Acanthamoeba causes diseases such as Acanthamoeba keratitis (AK) which leads to permanent blindness and granulomatous Acanthamoeba encephalitis (GAE) where there is formation of granulomas in the brain. Current treatments such as chlorhexidine, diamidines, and azoles either exhibit undesirable side effects or require immediate and prolonged treatment for the drug to be effective or prevent relapse. Previously, antifungal drugs amphotericin B, nystatin, and fluconazole-conjugated silver with nanoparticles have shown significantly increased activity against Acanthamoeba castellanii. In this study, two functionally diverse tetrazoles were synthesized, namely 5-(3-4-dimethoxyphenyl)-1H-tetrazole and 1-(3-methoxyphenyl)-5-phenoxy-1H-tetrazole, denoted by T1 and T2 respectively. These compounds were evaluated for anti-Acanthamoeba effects at different concentrations ranging from 5 to 50 μM. Furthermore, these compounds were conjugated with silver nanoparticles (AgNPs) to enhance their efficacy. Particle size analysis showed that T1-AgNPs and T2-AgNPs had an average size of 52 and 70 nm respectively. After the successful synthesis and characterization of tetrazoles and tetrazole-conjugated AgNPs, they were subjected to anti-Acanthamoeba studies. Amoebicidal assay showed that at concentration 10 μM and above, T2 showed promising antiamoebic activities between the two compounds while encystation and excystation assays reveal that both T1 and T2 have inhibited differentiation activity against Acanthamoeba castellanii. Conjugation of T1 and T2 to AgNP also increased efficacy of tetrazoles as anti-Acanthamoeba agents. This may be due to the increased bioavailability as AgNP allows better delivery of treatment compounds to A. castellanii. Human cell cytotoxicity assay revealed that tetrazoles and AgNPs are significantly less toxic towards human cells compared with chlorhexidine which is known to cause undesirable side effects. Cytopathogenicity assay also revealed that T2 conjugated with AgNPs significantly reduced cytopathogenicity of A. castellanii compared with T2 alone, suggesting that T2-conjugated AgNP is an effective and safe anti-Acanthamoeba agent. The use of a synthetic azole compound conjugated with AgNPs can be an alternative strategy for drug development against A. castellanii. However, mechanistic and in vivo studies are needed to explore further translational values.