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

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

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.

Establishment of an Acanthamoeba keratitis mouse model confirmed by amoebic DNA amplification.

Acanthamoeba castellanii, the causative agent of Acanthamoeba keratitis (AK), occurs mainly in contact lens users with poor eye hygiene. The findings of many in vitro studies of AK, as well as the testing of therapeutic drugs, need validation in in vivo experiments. BALB/c mice were used in this study to establish in vivo AK model. A. castellanii cell suspensions (equal mixtures of trophozoites and cysts) were loaded onto 2-mm contact lens pieces and inserted into mouse eyes that were scratched using an ophthalmic surgical blade under anesthesia and the eyelids of the mice were sutured. The AK signs were grossly observed and PCR was performed using P-FLA primers to amplify the Acanthamoeba 18S-rRNA gene from mouse ocular tissue. The experimental AK mouse model was characterized by typical hazy blurring and melting of the mouse cornea established on day 1 post-inoculation. AK was induced with at least 0.3 × 105 A. castellanii cells (optimal number, 5 × 104), and the infection persisted for two months. The PCR products amplified from the extracted mouse eye DNA confirmed the development of Acanthamoeba-induced keratitis during the infection periods. In conclusion, the present AK mouse model may serve as an important in vivo model for the development of various therapeutic drugs against AK.

Design, synthesis and antiamoebic activity of dysprosium-based nanoparticles using contact lenses as carriers against Acanthamoeba sp.

PURPOSE: Contact lenses have direct contact with the corneal surface and can induce sight-threatening infection of the cornea known as Acanthamoeba keratitis. The objective of this study was to evaluate the dysprosium-based nanoparticles (Dy-based NPs), namely Fe3 O4 -PEG-Dy2 O3 nanocomposites and Dy(OH)3 nanorods, as an active component against Acanthamoeba sp., as well as the possibility of their loading onto contact lenses as the drug administering vehicle to treat Acanthamoeba keratitis (AK). METHODS: The Dy-based NPs were synthesized, and they were loaded onto commercial contact lenses. The loading content of the NPs and their release kinetics was determined based on the absorbance of their colloidal solution before and after soaking the contact lenses. The cytotoxicity of the NPs was evaluated, and the IC50 values of their antiamoebic activity against Acanthamoeba sp. were determined by MTT colorimetric assay, followed by observation on the morphological changes by using light microscopy. The mechanism of action of the Dy-based NPs against Acanthamoeba sp. was evaluated by DNA laddering assays. RESULTS: The loading efficiencies of the Dy-based NPs onto the contact lens were in the range of 30.6-36.1% with respect to their initial concentration (0.5 mg ml-1 ). The Dy NPs were released with the flux approximately 5.5-11 µg cm-2  hr-1 , and the release was completed within 10 hr. The emission of the NPs consistently showed a peak at 575 nm due to Dy3+ ion, offering the possible monitoring and tracking of the NPs. The SEM images indicated the NPs are aggregated on the surface of the contact lenses. The DNA ladder assay suggested that the cells underwent DNA fragmentation, and the cell death was due most probably to necrosis, rather than apoptosis. The cytotoxicity assay of Acanthamoeba sp. suggested that Fe3 O4 -PEG, Fe3 O4 -PEG-Dy2 O3 , Dy(NO3 )3 .6H2 O and Dy(OH)3 NPs have an antiamoebic activity with the IC50 value being 4.5, 5.0, 9.5 and 22.5 µg ml-1 , respectively. CONCLUSIONS: Overall findings in this study suggested that the Dy-based NPs can be considered as active antiamoebic agents and possess the potential as drugs against Acanthamoeba sp. The NPs could be loaded onto the contact lenses; thus, they can be potentially utilized to treat Acanthamoeba keratitis (AK).

In Vivo Confocal Microscopy Morphologic Features and Cyst Density in Acanthamoeba Keratitis.

PURPOSE: To correlate in vivo confocal microscopy morphologic features (IVCM-MF) and Acanthamoeba cyst density (ACD) with final best-corrected visual acuity (BCVA) in Acanthamoeba keratitis (AK). DESIGN: Retrospective cohort study. METHODS: Patient demographics, treatment outcome, and corresponding IVCM-MF performed at the acute stage of infection were analyzed. Inclusion criteria were microbiological positive AK cases seen at Moorfields Eye Hospital between February 2013 and October 2017. Statistical significance was assessed by multinomial regression and multiple linear regression analysis. Main outcome measure was final BCVA. RESULTS: A total of 157 eyes (157 patients) had AK. Absence of single-file round/ovoid objects was associated with a BCVA of 6/36 to 6/9 (odds ratio [OR] 8.13; 95% confidence interval [CI], 1.55-42.56, P = .013) and ≥6/6 (OR 10.50; 95% CI, 2.12-51.92, P = .004) when compared to no perception of light to 6/60. Absence of rod/spindle objects was associated with a BCVA of ≥6/6 (OR 4.55; 95% CI, 1.01-20.45, P = .048). Deep stromal/ring infiltrate was associated with single-file round/ovoid objects (OR 7.78; 95% CI, 2.69-22.35, P < .001), rod/spindle objects (OR 7.05; 95% CI, 2.11-23.59, P = .002), and binary round/ovoid objects (OR 3.45; 95% CI, 1.17-10.14, P = .024). There was a positive association between ACD and treatment duration (ß = 0.14, P = .049), number of IVCM-MF (ß = 0.34, P = .021), and clusters of round/ovoid objects (ß = 0.29, P = .002). CONCLUSIONS: Specific IVCM-MF correlate with ACD and clinical staging of disease, and are prognostic indicators for a poorer visual outcome.

Corticosteroid eye drop instillation aggravates the development of Acanthamoeba keratitis in rabbit corneas inoculated with Acanthamoeba and bacteria.

The role of topical corticosteroids in management of Acanthamoeba keratitis (AK) remains controversial. Using a rabbit AK model, we investigated whether corticosteroid use is a risk factor of AK. Acanthamoeba (1 × 105/ml) was incubated with two densities of P. aeruginosa (PA; high-PA: 1 × 108/ml, low-PA: 3 × 105/ml) before corneal inoculation. Rabbit corneas were inoculated with Acanthamoeba alone or Acanthamoeba plus PA and administered levofloxacin and betamethasone sodium phosphate (BSP) eye drops for 5 or 7 days. Infected rabbit eyes were evaluated for clinical score and Acanthamoeba by histological examination. Acanthamoeba alone and BSP treatment did not produce keratitis. Corneas inoculated with Acanthamoeba plus low-PA treated immediately with levofloxacin and BSP remained clear with few infiltrates. Corneas inoculated with Acanthamoeba plus low-PA treated with levofloxacin immediately and BSP 12 h later developed severe keratitis. Corneas inoculated with Acanthamoeba plus high-PA treated immediately with levofloxacin and BSP also developed severe keratitis. Acanthamoebae were detected by PAS staining in corneas inoculated with Acanthamoeba plus high-PA treated with levofloxacin and BSP. Topical corticosteroids have the potential to aggravate AK when cornea is infected by Acanthamoeba with a critical number of bacteria or when corticosteroids are given after infection has established by Acanthamoeba with small number of bacteria.

N-chlorotaurine Inactivates Acanthamoeba and Candida albicans in the Porcine Ex Vivo Corneal Infection Model.

PURPOSE: N-chlorotaurine (NCT) is an anti-infective belonging to the class of chloramines and an investigative drug for the topical treatment of keratoconjunctivitis. The aim of the present study was to demonstrate its efficacy against Acanthamoeba and Candida in corneas infected ex vivo. METHODS: Corneal buttons from porcine eyes were contaminated with Acanthamoeba castellanii trophozoites or Candida albicans Centraalbureau voor Schimmelcultures 5982 and incubated for 7 and 3 days, respectively. Subsequently, they were treated with 1% NCT for 5 to 120 minutes. After further incubation for 2 days in the absence of NCT in tests with A. castellanii, the buttons were homogenized, and the amoebae grown for a further 5 days before they were counted in a light microscope. For C. albicans, quantitative cultures were performed from corneal homogenates. RESULTS: Incubation of 120 minutes in NCT completely inhibited the regrowth of A. castellanii and reduced the number of C. albicans colony-forming unit counts by 4 log10. In addition, at 60 minutes, significant reductions of both pathogens could be observed. Histology showed penetration of pathogens into the stroma of the corneal buttons. CONCLUSIONS: NCT inactivates A. castellanii and C. albicans in corneal tissue.

The Acanthamoeba-Fungal Keratitis Study.

PURPOSE: To ascertain the incidence of Acanthamoeba keratitis and the coexistence of Acanthamoeba and fungi in microbial keratitis. DESIGN: Prospective cross-sectional study. METHODS: Patients presenting with stromal keratitis were additionally tested for Acanthamoeba irrespective of the clinical diagnosis. Culture positivity was the gold standard. RESULTS: Of the 401 cases included in the study, 40 were positive for Acanthamoeba (10%); of these 40, 16 were positive for both Acanthamoeba and fungi (4.5% of the study group was Acanthamoeba and fungal keratitis positive); 5 were positive for Acanthamoeba and bacteria; and 2 had triple infection with Acanthamoeba, fungi, and bacteria. Ring infiltrates and stromal edema are frequently associated with Acanthamoeba keratitis, as well as in Acanthamoeba coinfections. Ring infiltrates in particular were more frequently seen in the Acanthamoeba and fungal keratitis group (8/16) and they were often yellowish with hyphate edges (vs ring infiltrates only, which are seen in the patients with Acanthamoeba alone). Only 2 patients were contact lens wearers: however, they presented with history of trauma. CONCLUSIONS: Acanthamoeba coinfections are much more frequent and are not restricted to contact lens users. Anticipating coinfections is necessary for establishing a diagnosis as well as for appropriate and timely therapeutic interventions.

Effect of Riboflavin/Rose Bengal-Mediated PACK-CXL on Acanthamoeba Trophozoites and Cysts in Vitro.

PURPOSE: To evaluate the antiamoebic properties of photo-activated chromophore for keratitis (PACK)-corneal cross-linking (CXL) (PACK-CXL), in combination with riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%), for treatment of Acanthamoeba trophozoites and cysts. MATERIALS AND METHODS: Cultures of Acanthamoeba castellanii were grown in a fluid medium at a concentration of 2.7 × 105 cell/ml. PACK-CXL was used on A. castellani cells in combination with either riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%). Riboflavin-containing wells were irradiated with ultraviolet-A (UVA) light (365-nm wavelength). Rose bengal-containing wells were irradiated with green light (523-nm wavelength). A power density of 9 mW/cm2 for 10 min and total irradiation dose of 5.4 J/cm2 was used for both riboflavin and rose bengal. After UVA and green light irradiation, cell viabilities were evaluated, and percentage of dead cells calculated. RESULTS: No significant amoebicidal activity was observed following PACK-CXL/riboflavin at either concentration. PACK-CXL/rose bengal, however, was observed to be highly effective in eradicating Acanthamoeba cells at either concentration, with no significant difference observed between the two concentrations. The percentage of dead cells was 63% following treatment at either rose bengal concentration. CONCLUSION: PACK-CXL with rose bengal demonstrated pronounced antiamoebic activity against A.castellanii. Further in vitro and in vivo studies are needed to confirm this finding.

Comparison of In Vivo Confocal Microscopy, PCR and Culture of Corneal Scrapes in the Diagnosis of Acanthamoeba Keratitis.

PURPOSE: Acanthamoeba keratitis (AK) is an uncommon but serious corneal infection, in which delayed diagnosis carries a poor prognosis. Conventional culture requires a long incubation period and has low sensitivity. Polymerase chain reaction (PCR) and in vivo confocal microscopy (IVCM) are available alternative diagnostic modalities that have increasing clinical utility. This study compares confocal microscopy, PCR, and corneal scrape culture in the early diagnosis of AK. METHODS: We reviewed the case notes of patients with a differential diagnosis of AK between June 2016 and February 2017 at the Bristol Eye Hospital, United Kingdom. Clinical features at presentation, and results of IVCM, PCR, and corneal scrape cultures were analyzed. RESULTS: A total of 25 case records were reviewed. AK was diagnosed in 14 patients (15 eyes). Based on the definition of "definite AK," the diagnostic sensitivities of IVCM, PCR, and corneal scrape cultures were 100% [95% confidence interval (CI), 63.1%-100%], 71.4% (95% CI, 41.9%-91.6%) and 33.3% (95% CI, 9.9%-65.1%), respectively. The 3 methods showed a specificity of 100% and a positive predictive value of 100%. Using a reference standard of only positive corneal cultures, IVCM, and PCR had a sensitivity of 100% (95% CI, 29.2%-100%) and 75% (95% CI, 19.4%-99.4%), respectively. CONCLUSIONS: All 3 diagnostic tests are highly specific, and a positive test result is highly predictive of disease presence. IVCM is both highly sensitive and specific when performed by an experienced operator. PCR is a useful adjunct in the diagnosis of AK because of its wider availability compared with IVCM, and it may be used in combination with IVCM for microbiologic confirmation.

Acanthamoeba keratitis in patients wearing scleral contact lenses.

PURPOSE: To report a series of cases of Acanthamoeba keratitis (AK) in scleral lens wearers with keratoconus to determine whether this type of contact lens presents a greater risk for development of infection. METHODS: This study reports three patients who wore scleral contact lenses to correct keratoconus and developed AK. The diagnoses of AK were established based on cultures of the cornea, scleral contact lenses, and contact lens paraphernalia. This study investigated the risk factors for infections. RESULTS: The possible risks for AK in scleral contact lens wearers are hypoxic changes in the corneal epithelium because of the large diameter and minimal tear exchange, use of large amounts of saline solution necessary for scleral lens fitting, storing the scleral lens overnight in saline solution rather than contact lens multipurpose solutions, not rubbing the contact lens during cleaning, and the space between the cornea and the back surface of the scleral lens that might serve as a fluid reservoir and environment for Acanthamoeba multiplication. Two patients responded well to medical treatment of AK; one is still being treated. CONCLUSIONS: The recommendations for use and care of scleral contact lenses should be emphasized, especially regarding use of sterile saline (preferably single use), attention to rubbing the lens during cleaning, cleaning of the plunger, and overnight storage in fresh contact lens multipurpose solutions without topping off the lens solution in the case.

Fibrous Catalyst-Enhanced Acanthamoeba Disinfection by Hydrogen Peroxide.

SIGNIFICANCE: Hydrogen peroxide (H2O2) disinfection systems are contact-lens-patient problem solvers. The current one-step, criterion-standard version has been widely used since the mid-1980s, without any significant improvement. This work identifies a potential next-generation, one-step H2O2, not based on the solution formulation but rather on a case-based peroxide catalyst. PURPOSE: One-step H2O2 systems are widely used for contact lens disinfection. However, antimicrobial efficacy can be limited because of the rapid neutralization of the peroxide from the catalytic component of the systems. We studied whether the addition of an iron-containing catalyst bound to a nonfunctional propylene:polyacryonitrile fabric matrix could enhance the antimicrobial efficacy of these one-step H2O2 systems. METHODS: Bausch + Lomb PeroxiClear and AOSept Plus (both based on 3% H2O2 with a platinum-neutralizing disc) were the test systems. These were tested with and without the presence of the catalyst fabric using Acanthamoeba cysts as the challenge organism. After 6 hours' disinfection, the number of viable cysts was determined. In other studies, the experiments were also conducted with biofilm formed by Stenotrophomonas maltophilia and Elizabethkingia meningoseptica bacteria. RESULTS: Both control systems gave approximately 1-log10 kill of Acanthamoeba cysts compared with 3.0-log10 kill in the presence of the catalyst (P < .001). In the biofilm studies, no viable bacteria were recovered following disinfection in the presence of the catalyst compared with ≥3.0-log10 kill when it was omitted. In 30 rounds' recurrent usage, the experiments, in which the AOSept Plus system was subjected to 30 rounds of H2O2 neutralization with or without the presence of catalytic fabric, showed no loss in enhanced biocidal efficacy of the material. The catalytic fabric was also shown to not retard or increase the rate of H2O2 neutralization. CONCLUSIONS: We have demonstrated the catalyst significantly increases the efficacy of one-step H2O2 disinfection systems using highly resistant Acanthamoeba cysts and bacterial biofilm. Incorporating the catalyst into the design of these one-step H2O2 disinfection systems could improve the antimicrobial efficacy and provide a greater margin of safety for contact lens users.

Nanoemulsions Containing a Coumarin-Rich Extract from Pterocaulon balansae (Asteraceae) for the Treatment of Ocular Acanthamoeba Keratitis.

This study describes the incorporation of a coumarin-rich extract from Pterocaulon balansae into nanoemulsions intended for the local treatment of ocular keratitis caused by Acanthamoeba. The n-hexane dewaxed extract of P. balansae was characterized by HPLC/PDA and UPLC/MS. The presence of four major coumarins was detected, where 5-methoxy-6,7-methylenedioxycoumarin was selected as a chemical marker. This extract was then incorporated into nanoemulsions composed of medium chain triglycerides and egg-lecithin, through spontaneous emulsification. Such a procedure yielded the formation of monodisperse nanoemulsions in a sub-300-nm range, regardless of the amount of extract incorporated (1.0-5.0 mg/mL). The amoebicidal activity against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent. A reduction of 95% of trophozoite viability was detected after 24 h of incubation with a nanoemulsion at 1.25 mg/mL of coumarins, being a similar effect detected for chlorhexidine. These results suggest a potential of the formulations developed in this study as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.

[Antibiotic susceptibility patterns of bacteria isolated from keratitis and intraocular infections at Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Colombia]. / Sensibilidad antibiótica de bacterias obtenidas de queratitis e infecciones intraoculares en la Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Colombia.

INTRODUCTION: Bacterial resistance is critical for the selection of antibiotics in the treatment of infections, so it is vital to know its current status in our geographical area. OBJECTIVE: To determine in vitro antibiotic susceptibility of bacterial isolates obtained from keratitis and intraocular infections. MATERIALS AND METHODS: A retrospective study of microbiological tests in Fundación Oftalmológica de Santander (FOSCAL) was carried out between June, 2011, and January, 2012. RESULTS: A total of 92 samples were examined and 110 bacteria, 27 fungi and 12 free-living amoebae were identified. Polymicrobial infections constituted 50% of the total; 1.1%, 0%, 1.1%, 16.9%, 29.3% and 85% of Gram-positive bacteria were resistant to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, respectively, while 0%, 8.3%, 0%, 0%, 18.2% and 27.3% of Gram-negative bacteria were resistant to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, respectively. For methicillin-resistant coagulase-positive staphylococci, resistance percentages to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin were 0%, 0%, 0%, 7%, 17% and 100%, respectively. For methicillin-resistant coagulase-negative staphylococci, resistance percentages to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin were 3%, 0%, 0%, 24%, 44% and 100%, respectively. Overall bacterial resistance to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, for both Gram-positive and Gram-negative, was 1%, 1%, 1%, 15.1%, 28% and 64.5%, respectively. CONCLUSIONS: The levels of bacterial resistance to imipenem, moxifloxacin and gatifloxacin were lower than for levofloxacin, ciprofloxacin and tobramycin. The levels of resistance to tobramycin were very high, which calls into question its usefulness in this region of our country.

Sensibilidad antibiótica de bacterias obtenidas de queratitis e infecciones intraoculares en la Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Colombia / Antibiotic susceptibility patterns of bacteria isolated from keratitis and intraocular infections at Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Colombia

Introducción. La resistencia bacteriana es crítica para la selección de los antibióticos en el tratamiento de las infecciones, por ello es vital conocer su estado actual en nuestro medio. Objetivo. Determinar la sensibilidad antibiótica bacteriana in vitro obtenida de los cultivos de queratitis e infecciones intraoculares. Materiales y métodos. Se llevó a cabo un estudio retrospectivo en la Fundación Oftalmológica de Santander (FOSCAL), entre junio de 2011 y enero de 2012. Resultados. Se examinaron 92 muestras. Se identificaron 110 bacterias, 27 hongos y 12 amebas de vida libre. Del total de bacterias Gram positivas, 1,1 %, 0 %, 1,1 %, 16,9 %, 29,3 % y 85 % fue resistente a imipenem, moxifloxacina, gatifloxacina, levofloxacina, ciprofloxacina y tobramicina, respectivamente, mientras que la resistencia a estos mismos fármacos se presentó, respectivamente, en 0 %, 8,3 %, 0 %, 0 %, 18,2 % y 27,3 % de las bacterias Gram negativas. Los porcentajes de resistencia de los estafilococos positivos para coagulasa resistentes a la meticilina fueron 0 %, 0 %, 0 %, 7 %, 17 % y 100 %, respectivamente, y los porcentajes de los estafilococos negativos para coagulasa resistentes a la meticilina fueron 3 %, 0 %, 0 %, 24 %, 44 % y 100 %, respectivamente. Los porcentajes de resistencia bacteriana globales (tanto para bacterias Gram positivas como para Gram negativas) a imipenem, moxifloxacina, gatifloxacina, levofloxacina, ciprofloxacina y tobramicina fueron 1 %, 1 %, 1 %, 15,1 %, 28 % y 64,5 %, respectivamente. Conclusiones. Los niveles de resistencia bacteriana para imipenem, moxifloxacina y gatifloxacina fueron menores que para levofloxacina, ciprofloxacina y tobramicina. Los niveles de resistencia para la tobramicina fueron muy altos, lo que pone en duda su utilidad clínica en las infecciones oculares en nuestro medio.

Introduction: Bacterial resistance is critical for the selection of antibiotics in the treatment of infections, so it is vital to know its current status in our geographical area. Objective: To determine in vitro antibiotic susceptibility of bacterial isolates obtained from keratitis and intraocular infections. Materials and methods: A retrospective study of microbiological tests in Fundación Oftalmológica de Santander (FOSCAL) was carried out between June, 2011, and January, 2012. Results: A total of 92 samples were examined and 110 bacteria, 27 fungi and 12 free-living amoebae were identified. Polymicrobial infections constituted 50% of the total; 1.1%, 0%, 1.1%, 16.9%, 29.3% and 85% of Gram-positive bacteria were resistant to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, respectively, while 0%, 8.3%, 0%, 0%, 18.2% and 27.3% of Gram-negative bacteria were resistant to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, respectively. For methicillin-resistant coagulase-positive staphylococci, resistance percentages to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin were 0%, 0%, 0%, 7%, 17% and 100%, respectively. For methicillin-resistant coagulase-negative staphylococci, resistance percentages to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin were 3%, 0%, 0%, 24%, 44% and 100%, respectively. Overall bacterial resistance to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, for both Gram-positive and Gram-negative, was 1%, 1%, 1%, 15.1%, 28% and 64.5%, respectively. Conclusions: The levels of bacterial resistance to imipenem, moxifloxacin and gatifloxacin were lower than for levofloxacin, ciprofloxacin and tobramycin. The levels of resistance to tobramycin were very high, which calls into question its usefulness in this region of our country.

Influence of Acanthamoeba genotype on clinical course and outcomes for patients with Acanthamoeba keratitis in Spain.

Genotype T4 is by far the most frequent genotype of Acanthamoeba keratitis (AK) and therefore has been considered the most virulent. This study included 14 cases of AK of genotype T4 and three cases of non-T4 genotype. We found that cases of non-T4 genotype had a worse response to medical therapy, greater need for surgical intervention, greater risk of extracorneal involvement, and remarkably poorer final visual outcome than those of T4 genotype, suggesting an association between Acanthamoeba virulence and genotype that requires additional case investigation.

Acanthamoeba keratitis: study of the 5-year incidence in Israel.

BACKGROUND: Acanthamoeba keratitis (AK) is not a notifiable disease in Israel, so there are no accurate incidence rates for this condition in Israel. The aim of this study was to estimate the incidence of AK in Israel for the years 2008-2012. METHODS: We distributed a survey questionnaire to laboratory managers in Israel. The laboratories were affiliated to medical institutes that either provided ophthalmology services or served community ophthalmology clinics. Our questionnaire requested survey respondents to provide information regarding the methods used to diagnose AK, and the number of positive and negative cultures for Acanthamoebae species performed for each of the years from 2008 to 2012. RESULTS: Six laboratories used non-nutrient agar with Escherichia coli as the culture medium, one used calcofluor-white staining with fluorescent microscopy, and two used PCR for diagnosing AK. Twenty-three AK cases were identified, to give an estimated incidence of 1/1 668 552. CONCLUSIONS: AK is mostly attributable to the use of contact lenses. As contact lenses are popular in Israel, we expected a higher incidence rate. A lower than expected incidence rate may indicate insufficient awareness of AK in Israel.

Coinfection with Acanthamoeba and Pseudomonas in contact lens-associated keratitis.

PURPOSE: To report coinfection with Acanthamoeba and Pseudomonas aeruginosa in a case with contact lens-associated keratitis. CASE REPORT: A 20-year-old woman presented to the emergency department of our hospital with a 4-day history of progressively increasing pain, redness, photophobia, mucopurulent discharge, and diminution of vision in her right eye. She was being treated for contact lens-related Pseudomonas keratitis in another hospital before presentation. Gram stain of corneal scrapings revealed gram-negative bacilli. Both Gram stain and 10% KOH wet mount showed the presence of Acanthamoeba cysts. Microbiological cultures obtained from contact lenses and contact lens storage case showed the presence of Pseudomonas aeruginosa and Acanthamoeba. Topical therapy was started in the form of hourly gentamycin 1.3%, cefazolin 5%, chlorhexidine 0.02%, propamidine 0.1%, polymyxin B 30,000 IU eye drops, and neosporin (neomycin, bacitracin, polymyxin) eye ointment four times a day. Symptomatic improvement was observed within 48 hours, along with a decrease in the density of infiltrates and a reduction in the anterior chamber reaction. Repeat corneal scrapings on day 10 showed Acanthamoeba but no bacilli. Progressive resolution of the infiltrate was noted during the next few days. Epithelialization was complete by day 24, following which the amoebicidal therapy was tapered during the next 4 weeks. Complete resolution of keratitis was achieved after 7 weeks of treatment. CONCLUSIONS: Both P. aeruginosa and Acanthamoeba are potentially devastating causes of microbial keratitis. Our case highlights the importance of considering the possibility of a concurrent infection in cases with contact lens-related keratitis.

Salicylate inhibition of acanthamoebal attachment to contact lenses.

PURPOSE: Sodium salicylate has shown potential as a component of contact lens care solutions designed to reduce Acanthamoebal attachment to contact lenses. This study determined the minimum effective concentration required to significantly reduce amoebal attachment. METHODS: Different concentrations of sodium salicylate (10, 15, and 20 mM) were applied during exposure of unworn or bacterial biofilm-coated hydrogel contact lenses to Acanthamoeba castellanii trophozoites. Salicylate was applied at stage 1 intervention during biofilm formation on lenses, at stage 2 intervention during amoebal exposure, or at both stages. RESULTS: A significant reduction in amoebal attachment was achieved when 10 mM salicylate was included during stage 1 alone; however, 15 mM was required for stage 2 intervention to significantly reduce attachment to clean or biofilm-coated lenses. For stages 1 and 2 combined intervention, 10 mM sodium salicylate produced a significant reduction in amoebal attachment. CONCLUSIONS: In situ, within a contact lens case, biofilm formation and amoebal attachment would occur concurrently; therefore, stages 1 and 2 intervention would be closest to the real-life situation, thus indicating that 10 mM of salicylate would be an effective minimum concentration for reducing amoebal attachment to hydrogel contact lenses. Inclusion of components in contact lens care solution, such as sodium salicylate, which reduce Acanthamoebal attachment, has the potential to enhance effectiveness, particularly where amoebicidal efficacy may be limited, thus reducing the risk of contact lens-associated Acanthamoebal infection.

Microbiological cure times in acanthamoeba keratitis.

AIMS: The purpose of this study was to estimate the duration of treatment necessary for sequential acanthamoeba laboratory tests from corneal scrapings to become negative, and to assess predictors that affect this duration period. METHODS: We included all patients with at least one positive acanthamoeba culture or Giemsa stain at the F.I. Proctor Foundation Microbiology Laboratory from 1996 to 2009. A parametric survival analysis was performed among patients with repeat cultures to assess significant predictors for extended clearance time. Simulations were performed to estimate clearance time in the entire patient population, assuming imperfect sensitivity. RESULTS: Thirty-seven patients with laboratory evidence of acanthamoeba had testing at 69 time points. The median clearance time among eyes with repeat cultures was 42.5 days (interquartile range (IQR) 22.0-82.0 days; unadjusted parametric model). Initial visual acuity was the only predictor significantly associated with clearance time in univariate analyses (P<0.0001). Using initial visual acuity as a predictor for clearance time among the entire patient population, the estimated clearance time decreased to 38.7 days (95% confidence interval (CI) 27.9-53.5 days). When the imperfect sensitivity of the culture technique was also taken into account, the estimated clearance time was 44.1 days (95% CI 31.9-61.0 days). CONCLUSION: The duration of infection with acanthamoeba keratitis undergoing treatment has not been well characterized. In this report we estimate a median clearance time of approximately 6 weeks, with an IQR of 22-82 days.