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

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

The global epidemiology and clinical diagnosis of Acanthamoeba keratitis.

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.

Experimental keratitis induced in rat by Acanthamoeba from distinct morphological groups/genotypes: a histological and immunohistochemical evaluation.

Species of the genus Acanthamoeba are free-living protozoans that occasionally act as parasites, causing a severe, progressive corneal infection termed Acanthamoeba keratitis (AK). The variable pathogenic potential among Acanthamoeba lineages has been shown by in vitro assays, but little is known about the behavior of different strains in animal models of AK. This work aimed to evaluate the infectivity of Acanthamoeba from distinct morphological groups and genotypes in a rat model of AK and apply an immunohistochemical technique for histological characterization of the lesions. Only a strain classified as group I/genotype T17, isolated from a soil source, caused ulcerated corneal lesions in two Wistar rats (n = 9) subjected to intrastromal inoculation. Two strains derived from AK human cases (group II/genotype T4 and group III/genotype T5) did not induce corneal lesions in the rats. A previous association of group II/genotype T4 trophozoites with lethally irradiated Escherichia coli did not influence the infectivity. A hyperimmune serum produced in Wistar rats was validated by an immunocytochemical technique using the three distinct strains and then applied for immunohistochemistry. The abundance of antigenic residues was observed in both corneas with keratitis, suggesting that the infectious process tended to resolve. Despite the low infection rate of the AK Wistar rat model, we produced an immunochemical tool with a potential diagnostic application. We also showed for the first time the ability of Acanthamoeba from T17 genotype to cause AK in experimental conditions.

Susceptibility of the Intact and Traumatized Feline Cornea to In Vitro Binding and Invasion by Acanthamoeba castellanii.

PURPOSE: Acanthamoeba castellanii ( A. castellanii ) displays host specificity at the level of the ocular surface. This study determined the susceptibility of the intact and traumatized feline cornea to A. castellanii binding and invasion relative to other host species with established susceptibility and resistance to Acanthamoeba binding. METHODS: Full-thickness buttons of fresh feline, porcine, and canine corneas were prepared. The corneal epithelium was confirmed intact by fluorescein staining or lightly scarified with a 25-G needle to simulate corneal trauma. Acanthamoeba castellanii was axenically cultivated. Corneal buttons were incubated with the parasite suspension or parasite-free medium for 18 hours at 35°C. Corneal buttons were rinsed, fixed, and processed for histopathology and immunohistochemistry using immunoperoxidase and immunofluorescence methods of amoeba detection. RESULTS: Numerous amoebae were bound to feline and porcine corneas incubated with parasites. In both intact and traumatized corneas, amoebae were detected at all levels in the corneal epithelium and within the anterior stroma. In traumatized corneal sections, amoebae were frequently present in regions of epithelial damage. Corneal architecture was well-preserved in sections incubated with parasite-free medium; however, epithelial cell sloughing, separation of epithelial layers, and epithelial detachment from the stroma were observed in corneas incubated with amoebae. Intact and traumatized canine corneas were relatively free of adherent amoebae, and corneal architecture was indistinguishable between sections incubated with the parasite suspension and parasite-free medium. CONCLUSIONS: The feline cornea is highly susceptible to in vitro binding and invasion by A. castellanii . Acanthamoeba binding to the feline cornea does not require a previous epithelial defect.

Amoebicidal Activity of Poly-Epsilon-Lysine Functionalized Hydrogels.

Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

Impact of Acanthamoeba Keratitis on the Vision-Related Quality of Life of Contact Lens Wearers.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Commensals Serve as Natural Barriers to Mammalian Cells during Acanthamoeba castellanii Invasion.

Acanthamoeba castellanii is a free-living, pathogenic ameba found in the soil and water. It invades the body through ulcerated skin, the nasal passages, and eyes and can cause blinding keratitis and granulomatous encephalitis. However, the mechanisms underlying the opportunistic pathogenesis of A. castellanii remain unclear. In this study, we observed that commensal bacteria significantly reduced the cytotoxicity of the ameba on mammalian cells. This effect occurred in the presence of both Gram-positive and Gram-negative commensals. Additionally, commensals mitigated the disruption of cell junctions. Ex vivo experiments on mouse eyeballs further showed that the commensals protected the corneal epithelial layer. Together, these findings indicate that A. castellanii is pathogenic to individuals with a dysbiosis of the microbiota at infection sites, further highlighting the role of commensals as a natural barrier during parasite invasion. IMPORTANCE Acanthamoeba castellanii, an opportunistic protozoan widely present in the environment, can cause Acanthamoeba keratitis and encephalitis in humans. However, only a few reports describe how the ameba acts as an opportunistic pathogen. Our study showed that the normal microbiota interfered with the cytotoxicity of Acanthamoeba, persevered during Acanthamoeba invasion, and reduced corneal epithelium peeling in the mouse eyeball model. This suggests that commensals may act as a natural barrier against Acanthamoeba invasion. In future, individuals who suffer from Acanthamoeba keratitis should be examined for microbiota absence or dysbiosis to reduce the incidence of Acanthamoeba infection in clinical settings.

Case Report: Molecular Detection and Characterization of Trypanosoma cruzi in Ocular Tissue from Donors with Chagas Disease.

Corneal transplantation is the most frequent transplant worldwide. Tissue characteristics allow storage and transport, even between continents, increasing its accessibility around the world. Donor infection with Trypanosoma cruzi is not defined as a corneal discarding factor, although the transplant is not recommended preventively, as in any infectious diseases. Herein, by means of polymerase chain reaction (PCR) strategies, we analyzed parasite presence in ocular tissue from 10 deceased donors with Chagas diseases. Among them, positive findings were obtained in corneas, scleras, and eye muscle samples of three, two, and one donor, respectively. Moreover, among the six T. cruzi defined populations, TcV and TcVI parasites were found in some samples based on group-specific amplification strategies. Our findings point out the actual possibility of T. cruzi transmission due to corneal transplantation and makes donor's serological status knowledge mandatory regardless of graft provenance. Failing that, we suggest a posttransplant follow-up of recipients from seropositive donors.

Detection of free-living amoebae in domestic cats with and without naturally-acquired keratitis.

Pathogenic free-living amoebae, most notably Acanthamoeba spp., are important pathogens of the human cornea. The importance of infection with free-living amoebae in cats with keratitis is currently unclear. The aim of this study was to determine the frequency of amoeba detection in corneas of cats with naturally-acquired keratitis and in the ocular surface microflora of cats without ocular disease. Clinical ophthalmic and in vivo corneal confocal microscopic examinations were performed on 60 cats with keratitis. Corneal scrapings were analyzed by amoeba culture; cytological evaluation; and Acanthamoeba, Hartmannella, and Vahlkampfia PCR assays. Following ophthalmic examination, conjunctival specimens collected from 60 cats without clinically apparent ocular disease were analyzed similarly. In one cat with ulcerative keratitis, amoeba cysts and trophozoites were detected by in vivo corneal confocal microscopy; an Acanthamoeba sp. was isolated from corneal specimens and detected by Acanthamoeba PCR assay; and suppurative corneal inflammation was present cytologically. An Acanthamoeba sp. was isolated from conjunctival specimens from one cat without clinically apparent ocular disease, but with suppurative inflammation demonstrated cytologically. Both Acanthamoeba isolates belonged to the T4 genotype. Naegleria-like amoebae were isolated in samples from two cats with keratitis and seven cats without clinical ocular disease, but amoebae were not detected by the other assays in these samples. Amoeba detection by culture was significantly (P = 0.01) associated with cytologically diagnosed corneoconjunctival inflammation. This study identified naturally-acquired Acanthamoeba keratitis in cats. Detection of Naegleria-like amoebae in samples from cats with and without keratitis is of uncertain pathological significance.

Delayed diagnoses of Acanthamoeba keratitis at a tertiary care medical centre.

PURPOSE: To determine the prevalence and reasons for delays in diagnosis in patients with Acanthamoeba keratitis (AK) presenting to Wilmer Eye Institute, Baltimore, Maryland. METHODS: This retrospective study analysed all patients with culture-positive AK seen between 2012 and 2019 at a tertiary referral centre. Patient demographic information, clinical history, risk factors, symptom duration, referral patterns, slit lamp examination findings, visual acuity and need for surgery were collected. RESULTS: The study included 45 eyes of 43 patients. On average, patients were symptomatic for 52.6 days before culture collection. Thirty-one percent of patients were diagnosed within 28 days of symptom onset while 69% were diagnosed after 28 days. Before presentation to a tertiary care centre, 69% of patients were evaluated by an ophthalmologist outside of this institution and 27% were evaluated by a provider other than an ophthalmologist. AK was most commonly misdiagnosed as herpetic keratitis, occurring in 38% of patients. The strongest risk factor for AK was contact lens use. Only 11% of patients presented with the classic ring infiltrate and 82% had pain. Patients with an early versus late diagnosis had a mean Snellen visual acuity (VA) of 20/224 versus 20/296 at presentation (p = 0.33) and a mean Snellen VA of 20/91 versus 20/240 at final visit (p = 0.07). 11% of patients required a therapeutic penetrating keratoplasty. CONCLUSION: Delayed diagnosis of AK in our cohort occurred due to a misdiagnosis as herpetic keratitis, non-specific clinical signs including the lack of pain in a number of patients, and a delay in referral to a tertiary care centre. Any contact lens wearer with an atypical keratitis should be referred promptly for Acanthamoeba cultures.

Microbiological diagnosis of Acanthamoebic keratitis: experience from tertiary care center of North India.

Acanthamoeba keratitis (AK) is a painful vision-threatening infection caused by pathogenic free-living Acanthamoeba. Due to the non-specific clinical presentation, this condition tends to be misdiagnosed by clinicians. A timely diagnosis is crucial for favorable visual outcome. Three hundred patients with suspected microbial keratitis presenting to the Advanced Eye Center at our tertiary care center in North India during the period from 2014 to 2018 were included. Patient's corneal scrapings, contact lens, lens solution, lens case, and tears were processed for microscopic examination by Giemsa and Calcofluor staining, non-nutrient agar (NNA) culture and molecular diagnosis by conventional PCR (cPCR) and Real-time PCR (qPCR). 18S rDNA gene sequencing was done to assess phylogenetic relationship. AK was found in 3.6% (11/300) of non-bacterial non-fungal keratitis patients. Among microbiological techniques, microscopy for Acanthamoeba was positive in 7 cases, NNA culture was positive in 9 cases and 11 cases were detected both by cPCR and qPCR. The sensitivity of microscopy, culture, cPCR and qPCR was 63.64%, 81.82 %, 100%, and 100% respectively whereas specificity was 100% for all the tests. 18S rDNA sequencing revealed that A. castellanii was the predominant species and isolates were genetically distinct. AK should be considered in the differential diagnosis of infectious keratitis. Molecular tests are useful for rapid, sensitive and specific diagnosis and must be included in workup of keratitis.

Resolution of Acanthamoeba Keratitis with Adjunctive Use of Oral Miltefosine.

Purpose: To report a series of cases demonstrating the resolution of Acanthamoeba keratitis (AK) with adjunctive use of oral miltefosine.Methods: Retrospective case series.Results: The first case was a 27-year-old female who presented with severe pain and photophobia. The diagnosis of AK was made with confocal microscopy, which revealed a significant burden of stromal cysts. After approximately 2 weeks of adjunctive oral miltefosine therapy, there was a severe inflammatory response within the cornea followed by quick resolution of the AK. The second case was a 31-year-old male in whom the diagnosis of AK was confirmed by culture and polymerase chain reaction. Adjunctive oral miltefosine was started 3 months after presentation, leading to a quick resolution.Conclusions: Oral miltefosine may have cysticidal properties and should be considered as adjunctive therapy for the treatment of AK, particularly in cases with a significant burden of cysts or in cases recalcitrant to other treatments.

The biology of Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a rare protozoal infection of the cornea. At least eight species of Acanthamoeba are known to cause this sight-threatening disease of the ocular surface. Acanthamoeba spp. exist in a wide array of niches ranging from thermal springs to under ice and every conceivable habitat in between. Contact lens wear is the leading risk factor for AK and is practiced by over 30 million individuals in the United States, yet the incidence of AK is less than 33 cases per one million contact lens wearers. Serological studies have reported that 90%-100% of individuals with no history of AK possess antibodies specific for Acanthamoeba antigens indicating that exposure to this organism is commonplace, yet disease is remarkably rare. Animal studies have shed light on the pathobiology and immunobiology of AK and indicate that a constellation of factors including the ocular surface microbiome and the microbiome of Acanthamoeba itself contribute to the pathogenesis of AK. Interesting, secretory antibodies produced by the adaptive immune response can prevent the initiation of corneal infection, but once Acanthamoeba trophozoites breach the corneal epithelium the adaptive immune system is helpless in altering the course of AK. It has been almost 50 years since AK was first described, yet many questions remain unanswered about this curious and enigmatic disease of the ocular surface.

Linezolid shows high safety and efficacy in the treatment of Pythium insidiosum keratitis in a rabbit model.

Efficacy and safety of three antibiotics (Linezolid-LZ, 0.2%; Azithromycin-AZ, 1%; Tigecycline-TG, 1%) were determined in the treatment of Pythium insidiosum keratitis in rabbits. Infection of right eye of 38 rabbits was induced by standard intracorneal injection of P. insidiosum zoospores (left eye, intracorneal saline). Corneal infection developed in all right eyes. One hourly eye drops of one of the three antibiotics was instilled in both eyes (3 groups of 12 rabbits each) except in controls. Half of the rabbits in each group received intracorneal injection of the respective antibiotic after 4 days of starting eye drops. Clinical scoring of eyes was done over next 3 weeks. The reduction in scores post-treatment was significant for each drug (LZ: p < 0.025, AZ: p < 0.025, TG: p < 0.01). Scores with LZ (median change of 3) was significantly (p = 0.013) higher than TG (median change of 2) and comparable (p = 0.06) to AZ (median change of 3). Reduction in clinical scores in eyes receiving intracorneal antibiotics was not significantly different from the eyes that did not receive intracorneal antibiotics (p = 0.73). While no adverse effect of LZ was seen in the control corneas, 66-100% of rabbits showed reaction to AZ and TG. Histopathology showed severe inflammation in all infected corneas and intraocular extension in some of the rabbits with poor response. The success rate was noted to be 16.7%, 25% and 50% in AZ, TG and LZ respectively (p = 0.45). LZ demonstrated superior efficacy and safety and can be considered for trial in human disease.

Ocular complications of oak processionary caterpillar setae in the Netherlands; case series, literature overview, national survey and treatment advice.

During early summer 2019, the Netherlands experienced an outbreak of the exotic oak processionary caterpillar. The vast number of caterpillars, which live in large nests on oak trees before they turn into moths, possess thousands of small, barbed hairs (setae) that are disseminated with the wind. The hairs cause a range of primarily dermatologic problems. However, Dutch ophthalmologists started reporting patients with ophthalmologic complaints caused by the penetrating hairs of the oak processionary caterpillar. This paper focuses on the ophthalmologic complications caused by the caterpillar hairs. We collected a series of four cases with reports ranging from a corneal erosion with hairs lodged into the cornea, to a sterile endophthalmitis in which hairs were found in the vitreous. A literature review for similar cases was performed using the PubMed and Embase database. Together with the Dutch Ophthalmic Society (Nederlands Oogheelkundig Gezelschap, NOG), a national survey was issued to determine the scale of this new problem. This showed that oak processionary caterpillar related complaints are primarily limited to the south of the Netherlands. Suggested ophthalmic treatment guidelines are presented. With the next summer at the doorstep, and limited preventative measures against the caterpillar hairs, we expect a new wave of ophthalmologic complaints coming year as well.

Cluster of Post-Operative Endophthalmitis Caused by Acanthamoeba T10 Genotype - A First Report.

PURPOSE: To report a cluster of postoperative Acanthamoeba endophthalmitis after routine cataract surgeries. METHODS: A brief summary of sentinel events leading to the referral of 4 patients of postoperative endophthalmitis to our hospital is followed by clinical descriptions and the various diagnostic approaches and interventions used. Genotyping and phylogenetic analysis are also discussed. RESULTS: Four cases of postoperative cluster endophthalmitis, presumed to be bacterial and treated as such, were referred to our hospital. The presence of an atypical ring infiltrate in the first case facilitated the diagnosis of Acanthamoeba endophthalmitis. All patients had vitritis, corneal involvement, and scleral inflammation. Multiple diagnostic methods, such as corneal scrapings, confocal microscopy, aqueous and vitreous taps, scleral abscess drainage, histopathological studies, polymerase chain reaction, and genotyping and phylogenetic analyses of isolated Acanthamoeba, were used to confirm the diagnosis of endophthalmitis and to establish the extent of ocular involvement. Various medical and therapeutic interventions used to control the infections were also documented. The isolated Acanthamoeba were confirmed as belonging to the T10 genotype, an environmentally and clinically rare variety. CONCLUSIONS: This is the first report of a cluster of postoperative T10 genotype Acanthamoeba endophthalmitis, occurring after routine cataract surgery in immunocompetent individuals. Contrary to current perceptions, a rapidly evolving infection can occur with Acanthamoeba.

PCR and culture for diagnosis of Acanthamoeba keratitis.

BACKGROUND/AIMS: Acanthamoeba 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. METHODS: 1217 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. RESULTS: Estimated 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%). CONCLUSIONS: Culture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

Acanthamoeba Keratitis Versus Mixed Acanthamoeba and Bacterial Keratitis: Comparison of Clinical and Microbiological Profiles.

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.