Fabio Ramos de Souza Carvalho

Twenty years of acanthamoeba keratitis

Purpose: We described the rate of Acanthamoeba keratitis (AK) in a referral eye center in São Paulo, Brazil, through a retrospective review of clinical and laboratorial records of patients over 2 decades. Methods: From 1987 to 2006, a total of 581 requests for amoebic laboratory workup in cases of infectious keratitis were investigated. Statistical analyses were applied to analyze a tendency of AK cases. Results: Acanthamoeba species were cultured from corneal scrapings of 185 patients, 5 of them with bilateral infection. Eighty-three percent of those patients were related with contact lens wear. Conclusions: The results suggested that patients with AK have persisted and increased over time at our ophthalmology center. Contact lenses showed to be a potential risk factor. Amoebic corneal infection can be considered as a new but well-established disease in Brazilian ophthalmology and visual sciences.

Assessing Efficacy of Combined Riboflavin and UV-A Light (365 nm) Treatment of Acanthamoeba Trophozoites

PURPOSE To assess the Acanthamoeba trophozoite viability in vitro and treatment of Acanthamoeba keratitis in a hamster model using ultraviolet light A (UV-A) and riboflavin (B2). METHODS A sample of Acanthamoeba sp. cultured was transferred to a 96-well plate and exposed to B2 and the UV-A light (365 nm wavelength) at a power density of 3 mW/cm(2), 8 mm spot diameter, for 30 minutes. The exposure was done in triplicate. Control groups were prepared in triplicate as well: blank control, UV-A only, riboflavin only, and dead control. Cell viability assessment was done using the trypan blue dye exclusion method. Acanthamoeba keratitis was induced in Chinese hamsters; who were randomly assigned to one of the animal groups: UV-A + B2, propamidine isethionate (Brolene; Sanofi-Aventis, Ellerslie, Auckland, Australia), UV-A + B2 + propamidine isethionate (Brolene), only UV-A, only B2, and blank. Throughout the 14 days after treatment the animals were examined clinically. Histology and clinical scores of all groups were compared. RESULTS The in vitro study showed no difference between the treatment group UV-A + B2 and the control groups. In the hamster keratitis model a significant improvement of clinical score was observed for the groups propamidine isethionate (Brolene) and UV-A + B2 + propamidine isethionate (Brolene) (P = 0.0067). Also a significant worsening of clinical score was observed in the other groups: UV-A + B2 group (P = 0.0084), only UV-A (P = 0.0078), B2 only (P = 0.0084), and blank (P = 0.0082). No difference was observed between propamidine isethionate (Brolene) and UV-A + B2 + propamidine isethionate (Brolene). CONCLUSIONS The adjunctive use of UV-A and B2 therapy did not demonstrate antitrophozoite activity; in vivo UV-A and B2 did not demonstrate efficacy in this model.

Therapeutic agents and biocides for ocular infections by free-living amoebae of Acanthamoeba genus

Acanthamoeba keratitis is a sight-threatening infectious disease. Resistance of the cystic form of the protozoan to biocides and the potential toxicity of chemical compounds to corneal cells are the main concerns related to long-term treatment with the clinically available ophthalmic drugs. Currently, a limited number of recognized antimicrobial agents are available to treat ocular amoebic infections. Topical application of biguanide and diamidine antiseptic solutions is the first-line therapy. We consider the current challenges when treating Acanthamoeba keratitis and review the chemical properties, toxicities, and mechanisms of action of the available biocides. Antimicrobial therapy using anti-inflammatory drugs is controversial, and aspects related to this topic are discussed. Finally, we offer our perspective on potential improvement of the effectiveness and safety of therapeutic profiles, with the focus on the quality of life and the advancement of individualized medicine.

Anxiety-like effects of meta-chlorophenylpiperazine in paradoxically sleep-deprived mice

Chlorophenylpiperazines (CPP) are psychotropic drugs used in nightclub parties and are frequently used in a state of sleep deprivation, a condition which can potentiate the effects of psychoactive drugs. This study aimed to investigate the effects of sleep deprivation and sleep rebound (RB) on anxiety-like measures in mCPP-treated mice using the open field test. We first optimized our procedure by performing dose-effect curves and examining different pretreatment times in naïve male Swiss mice. Subsequently, a separate cohort of mice underwent paradoxical sleep deprivation (PSD) for 24 or 48h. In the last experiment, immediately after the 24h-PSD period, mice received an injection of saline or mCPP, but their general activity was quantified in the open field only after the RB period (24 or 48h). The dose of 5mgmL(-1) of mCPP was the most effective at decreasing rearing behavior, with peak effects 15min after injection. PSD decreased locomotion and rearing behaviors, thereby inhibiting a further impairment induced by mCPP. Plasma concentrations of mCPP were significantly higher in PSD 48h animals compared to the non-PSD control group. Twenty-four hours of RB combined with mCPP administration produced a slight reduction in locomotion. Our results show that mCPP was able to significantly change the behavior of naïve, PSD, and RB mice. When combined with sleep deprivation, there was a higher availability of drug in plasma levels. Taken together, our results suggest that sleep loss can enhance the behavioral effects of the potent psychoactive drug, mCPP, even after a period of rebound sleep.

The Use of Botulinum Toxin to Treat Infantile Esotropia: A Systematic Review With Meta-Analysis

Purpose The purpose of this review was to examine the efficacy of botulinum toxin in the treatment of infantile esotropia and to evaluate the average response of BT and its complication rates. Methods A research was performed in the Latin American and Caribbean Literature on Health Sciences (LILACS), MEDLINE, and Cochrane Central Register of Controlled Trial (CENTRAL). The database was searched between December 28, 2016 and January 30, 2017. The selection was restricted to articles published in English, Spanish, or Portuguese. There were no date restrictions in the search. Results Nine studies were eligible for inclusion. The grouped success rate of BT treatment in infantile esotropia was 76% (95% confidence interval [CI]: 61%-89%). For the success rate, I2 of 94.25% was observed, indicating a high heterogeneity (P < 0.001). The complication rates were also analyzed. The grouped consecutive exotropia (XT) rate was 1% (95% CI: 0%-2%). The grouped ptosis rate was 27% (95% CI: 21%-33%). The grouped vertical deviation rate was 12% (95% CI: 4%-22%). The mean change of the deviation after BT injection was -30.7 (95% CI: -37.7, -23.8), demonstrating a significant improvement in alignment. Conclusions Botulinum toxin injection into medial recti muscles reveals to be a safe procedure and a valuable alternative to strabismus surgery in congenital esotropia, especially in moderate deviations.

Toxicity of Intracameral Injection of Fourth-Generation Fluoroquinolones on the Corneal Endothelium

Purpose: The aim of this study was to compare the cellular susceptibility patterns and morphologic changes in the corneal endothelium associated with the use of fourth-generation fluoroquinolones. Method: Endothelial susceptibility was assessed through intracameral injection of besifloxacin, gatifloxacin, and moxifloxacin. Human umbilical vein endothelial cells (HUVECs) were used as the standard cellular lineage to assess the quantitative toxicity of each antibiotic solution. Qualitative changes in the morphologic character of the corneal structure and the endothelial layer were generated using a combination of ex vivo and in vivo assays. Experimental assays were conducted in triplicate, and the results were statistically analyzed. Results: At 1 hour of exposure, all HUVECs exposed to antibiotics showed viability above 85%, after 3 hours of exposure to besifloxacin, gatifloxacin, and moxifloxacin, the percentages of viable cells were 68.3 ± 4.0 (P < 0.001), 90.7 ± 4.2 (P < 0.05), and 93.3 ± 1.5 (P > 0.05), respectively. All fluoroquinolones tested showed toxicity to HUVECs, resulting in significant (P < 0.001) loss of cellular viability after 24 hours of drug exposure. Giant endothelial cells were observed in animals treated with the 3 fluoroquinolones in contrast to the absence of these abnormal cells in the untreated group. Early cellular detachment was seen in the endothelial layer after exposure to gatifloxacin and moxifloxacin. Conclusions: We concluded that injection of fourth-generation fluoroquinolones in the aqueous humor did not adversely affect the corneal endothelium. However, these results suggested that prophylactic intracameral injection of besifloxacin, gatifloxacin, or moxifloxacin, if needed, should be administered as a last therapeutic resource in clinical practice, with careful and constant monitoring of corneal endothelium.

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.

Clinical Implications of Acanthamoeba Affinity for Electric Fields

Acanthamoeba keratitis (AK) has been most commonly related to careless contact lens (CL) handling and/or minor contaminated corneal abrasions. The treatment of AK has been characterized by the complexity of virulence and resistance profiles expressed by the protozoa in different layers of the corneal tissue and the toxicity of antimicrobial compounds used in the ophthalmologic routine for a long period of therapy. Furthermore, a delay in diagnosis may lead to the need for high-risk corneal transplantation and/or may result in eventual blindness. For these reasons, the establishment and development of preventive measures and procedures are of major importance in the control of the disease. The relevance of electrophysiology application in the ophthalmology and visual sciences fields, focused on main aspects of clinical and basic research on the corneal surface, has been reported. 1 In this issue of IOVS, Rudell and colleagues 2 elegantly show that Acanthamoeba trophozoites significantly respond to electric fields (EFs). The voltage dependence relation is also demonstrated. The authors are able to show the ability of trophozoites to migrate directionally toward the cathode; these results could lead to relevant further studies related to the application of electrophysiology in animal models of Acanthamoeba keratitis. Once the application of EFs induced the migration of trophozoites from deeper layers of cornea and consequently increased the exposure of trophozoites to antimicrobial compounds out of the corneal stroma, the researchers should feel encouraged to develop further studies in this field of science focused on in vivo assays related to the electrophysiology in Acanthamoeba keratitis. The findings could also provide a better understanding concerning the radial neuritis and/or corneal nerve alterations caused by the activation of inflammatory cascade due to the presence of Acanthamoeba trophozoites around the surface of corneal nerve, 3 which is negatively charged in nonmyelinated nerves during the process of membrane depolarization followed by electrical pulse. In summary, the article by Rudell and collaborators presents impressive scientific information to provide translational and reverse translational approaches concerning the electrophysiology of Acanthamoeba cysts and trophozoites. Furthermore, the findings described in the manuscript could open new perspectives for therapeutic approaches in Acanthamoeba keratitis.