Staurosporine as a Potential Treatment for Acanthamoeba Keratitis Using Mouse Cornea as an Ex Vivo Model.

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas.

Acanthamoeba castellanii: Effect of neuroactive substances on trophozoite migration.

Acanthamoeba castellanii is the etiological agent of granulomatous amebic encephalitis, amebic keratitis, and skin lesions. In vitro and in vivo studies have demonstrated that Acanthamoeba trophozoites induce contact-dependent, and contact-independent pathogenic mechanisms. We have explored the potential role neuroactive substances may have in the migration of Acanthamoeba castellanii trophozoites using Transwell permeable supports in the presence of physiological concentrations of dopamine, glutamate, serotonin, or taurine diluted in PBS. Quantitation of migrated amoebae was carried out in scanning electron micrographs of the upper and under compartments sides of the chamber membranes. Our results showed that at 2 h of interaction, a statistically significant larger proportion of A. castellanii trophozoites migrated through the chamber membranes when neurotransmitters were placed in the lower compartments of the chambers compared to control. This migration effect was more evident under the presence of glutamate and taurine on the three surfaces (upper/lower membrane and bottom compartment) when the percentage of migrated trophozoites was analyzed. Scanning electron microscopy of trophozoites revealed that glutamate and taurine induced the formation of large adhesion lamellas and phagocytic stomas. These observations suggest that certain neuroactive substances could stimulate the migration of A. castellanii trophozoites in the central nervous system.

Evaluation of the sensitivity to chlorhexidine, voriconazole and itraconazole of T4 genotype Acanthamoeba isolated from Mexico.

Free-living amoebae of the genus Acanthamoeba are the etiological agents of cutaneous lesions, granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK), which are chronic infections with poor prognosis if not diagnosed promptly. Currently, there is no optimal therapeutic scheme to eradicate the pathologies these protozoa cause. In this study we report the morphological and molecular identification of three species of the genus Acanthamoeba, belonging to T4 group; A. polyphaga isolated from the corneal ulcer of a patient sample of AK case; A. castellanii isolated from the contact lens of an AK patient and A. palestinensis obtained from a soil sample. The in vitro activity of chlorhexidine, itraconazole and voriconazole drugs against trophic stage was also evaluated through a colorimetric assay based on the oxidation-reduction of alamar blue. The strains in the study were sensitive to the evaluated drugs; although when determining the 50% inhibitory concentration (IC50) statistically significant differences were observed. A. castellanii showed to be highly sensitive to voriconazole (0.66 ±â€¯0.13 µM) but the least sensitive to chlorhexidine and itraconazole (8.61 ±â€¯1.63 and 20.14 ±â€¯4.93 µM, respectively), A. palestinensis showed the highest sensitivity to itraconazole (0.502 ±â€¯0.11 µM) and A. polyphaga expressed moderate sensitivity to chlorhexidine and itraconazole and lower sensitivity to voriconazole (10.10 ±â€¯2.21 µM). These results showed that species of the genus Acanthamoeba express different sensitivity to the tested drugs, which could explain the problems surrounding the establishment of a treatment of choice in the infections caused by these amoebae. We consider that although chlorhexidine and itraconazole show good activity on these amoebae and have been used in cases of AK in Mexico with acceptable results, voriconazole should be considered as the first therapeutic option of future Acanthamoeba infections that will be diagnosed in our country.

Acanthamoeba keratitis in Mexico: Report of a clinical case and importance of sensitivity assays for a better outcome.

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection. The early symptoms include redness, pain, photophobia and intense tearing. Chronic infection usually progresses to stromal inflammation, ring ulcers, corneal opacification and hypopyon. Here we document an AK case in a high myopic 38-year-old woman from Mexico City, with a history of wearing contact lenses while swimming. Corneal scrapes cultures were positive only for amoebae, consequently a treatment including netilmicin 0.3% and oral itraconazole 100 mg/12 h was prescribed. The infection was resolved after 8 months, leaving a slight leucoma outside the visual axis, with a visual acuity of 20/150. In the laboratory, the amoebic isolate was axenized in PYG medium, with an optimal growth at 30 °C, and was identified morphologically as Acanthamoeba polyphaga according to the taxonomic criteria of Page (1988) and placed in the T4 group by genotyping. The virulence of this strain (40%) was determined by intranasal inoculation of 1 × 106/20 µl trophozoites in BALB/c mice recovering from brain, proving their invasion ability and by the interaction with monolayers of epithelial cells of the established MDCK line of canine kidney origin (1:2 ratio of interaction), at 1, 3, 6, 8 and 24 h; trophozoites migrated to cell junctions inducing few lytic zones. In addition to the biological characterization, in vitro drug sensitivity tests were performed using chlorhexidine, itraconazole, netilmicin and voriconazole. Results revealed that voriconazole was the most effective compound. A. polyphaga remains as one of the most frequently isolated species producing AK. The treatment of AK case using netilmicin and oral itraconazole solved the disease, but the healing process was wide-ranging (8 months). The use of voriconazole and chlorhexidine may be an alternative treatment of future AK cases in Mexico.

Acanthamoeba culbertsoni isolated from a clinical case with intraocular dissemination: Structure and in vitro analysis of the interaction with hamster cornea and MDCK epithelial cell monolayers.

Acanthamoeba culbertsoni trophozoites, previously isolated from a human keratitis case with severe intraocular damage, were maintained in axenic culture. Co-incubation of amoebae with MDCK cell monolayers demonstrated an apparent preference of the amoebae to introduce themselves between the cells. The trophozoites appeared to cross the cell monolayer through the tight junctions, which resulted in decreased trans-epithelial resistance (TER) measurements. Unexpectedly, after co-incubation of amoebae with hamster corneas, we observed that the trophozoites were able to cross the different cell layers and reach the corneal stroma after only 12 h of interaction, in contrast to other Acanthamoeba species. These observations suggest that this A. culbertsoni isolate is particularly pathogenic. Further research with diverse methodologies needs to be performed to explain the unique behavior of this Acanthamoeba strain.

Type 2 diabetes mellitus BALB/c mice are more susceptible to granulomatous amoebic encephalitis: Immunohistochemical study.

Granulomatous amoebic encephalitis (GAE) is a chronic, difficult to resolve infection caused by amphizoic amoebae of the genus Acanthamoeba, which in most cases occurs in immunosuppressed persons or with chronic diseases such as diabetes. In this study, we describe the early events of A. culbertsoni infection of GAE in diabetic mice model. Diabetes was induced in male BALB/c mice, with a dose of streptozotocin (130 mg/kg). Healthy and diabetic mice were inoculated via intranasal with 1 × 106 trophozoites of A. culbertsoni. Then were sacrificed and fixed by perfusion at 24, 48, 72 and 96 h post-inoculation, the brains and nasopharyngeal meatus were processed to immunohistochemical analysis. Invasion of trophozoites in diabetic mice was significantly greater with respect to inoculated healthy mice. Trophozoites and scarce cysts were immunolocalized in respiratory epithelial adjacent bone tissue, olfactory nerve packets, Schwann cells and the epineurium base since early 24 h post-inoculation. After 48 h, trophozoites were observed in the respiratory epithelium, white matter of the brain, subcortical central cortex and nasopharyngeal associated lymphoid tissue (NALT). At 72 h, cysts and trophozoites were immunolocalized in the olfactory bulb with the presence of a low inflammatory infiltrate characterized by polymorphonuclear cells. Scarce amoebae were observed in the granular layer of the cerebellum without evidence of inflammation or tissue damage. No amoebas were observed at 96 h after inoculation, suggesting penetration to other tissues at this time. In line with this, no inflammatory infiltrate was observed in the surrounding tissues where the amoebae were immunolocalized, which could contribute to the rapid spread of infection, particularly in diabetic mice. All data suggest that trophozoites invade the tissues by separating the superficial cells, penetrating between the junctions without causing cytolytic effect in the adjacent cells and subsequently reaching the CNS, importantly, diabetes increases the susceptibility to amoebae infection, which could favor the GAE development.

Acanthamoeba (T4) trophozoites cross the MDCK epithelium without cell damage but increase paracellular permeability and transepithelial resistance by modifying tight junction composition.

Free-living amoebae of the genus Acanthamoeba are protozoa ubiquitously found in nature. Some species of the genus are potentially pathogenic for humans provoking keratitis in healthy individuals, often in contact lens wearers and opportunistic infections such as pneumonitis, fatal granulomatous encephalitis and skin infections, particularly in immunocompromised individuals. The pathogenic mechanisms of these amoebae are poorly understood, however it had been suggested that contact dependent mechanisms are important during invasion, regardless of the epithelia type, since amoebae penetrate epithelia separating tight junction (TJ). This study was undertaken to determine whether Acanthamoeba sp. (T4) damages the barrier function of the TJ in MDCK epithelial monolayers. Actin cytoskeleton staining and electron microscopy analyses were performed; paracellular permeability and TJ sealing were evaluated by apicobasolateral diffusion of ruthenium red and transepithelial resistance (TER) measurements; immunofluorescence and Western blot assays were performed to locate and estimate expression of TJ protein claudins 2 (Cldn2) and 4 (Cldn4). The results show that Acanthamoeba sp. crosses the MDCK monolayer without altering the actin cytoskeleton or the morphology of the cells. When trophozoites or conditioned medium interact with the monolayer, paracellular diffusion of ruthenium red increases. After 6 h, the amoebae, but not their conditioned medium, increase the TER, and Cldn2 is removed from the TJ, and its overall content in the cells diminishes, while Cldn4 is targeted to the TJ without changing its expression level. In conclusion Acanthamoeba (T4) crosses MDCK monolayer without damaging the cells, increasing permeability and TER through Cldn2 degradation, and redirecting Cldn4 to TJ. These results strongly suggest that contact-dependent mechanisms are relevant during amoebae invasion.

In vivo CNS infection model of Acanthamoeba genotype T4: the early stages of infection lack presence of host inflammatory response and are a slow and contact-dependent process.

This study was developed in order to describe the early morphological events observed during the invasion of two pathogenic strains of Acanthamoeba (genotype T4); A. castellanii and A. culbertsoni, at the olfactory meatus and cerebral, pulmonary, renal, hepatic and splenic tissues levels, an in vivo invasion study. Histological and immunohistochemical description of the events at 24, 48, 72, and 96 h postintranasal inoculations of BALB/c mice was performed. A. castellanii showed a higher invasion rate than A. culbertsoni, which was only able to reach lung and brain tissue in the in vivo model. The current study supports previous evidence of lack of inflammatory response during the early stages of infection. Acanthamoeba invasion of the CNS and other organs is a slow and contact-dependent process. The early morphological events during the invasion of amoebae include the penetration of trophozoites into different epithelia: olfactory, respiratory, alveolar space, and renal tubule, which resemble the process of amoebae invasion described in corneal tissue. The data suggest that after reaching the nasal epithelium, trophozoites continued invasion, separating and lifting the most superficial cells, then migrating and penetrating between the cell junctions without causing a cytolytic effect on adjacent cells. These results reaffirm the idea that contact-dependent mechanisms are relevant for amoebae of Acanthamoeba genus regardless of the invasion site.

Acanthamoeba culbertsoni: Electron-Dense Granules in a Highly Virulent Clinical Isolate.

The virulence of various amoebic parasites has been correlated with the presence of electron-dense granules (EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long-term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long-term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni.

Acanthamoeba genotypes T3 and T4 as causative agents of amoebic keratitis in Mexico.

Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.

Balamuthia mandrillaris: Further morphological observations of trophozoites by light, scanning and transmission electron microscopy.

Additional morphological features of Balamuthia mandrillaris observed by light and electron microscopy are reported. Trophozoites were extremely pleomorphic: their cell shapes ranged from rounded to elongated and sometimes they appeared exceptionally stretched out and branched. By transmission electron microscopy it was possible to observe two different cytoplasmic areas, the ectoplasm and the endoplasm and often sections of rough endoplasmic reticulum were found in the transition zone. The cytoplasm was very fibrogranular and most of the organelles typically found in eukaryotic cells were observed. A particular finding was the presence of numerous mitochondria with a different structure from those of other free-living amoebae. The observations reported here may reinforce the morphological knowledge of this amoeba and provide a background for further analyses.

Naegleria fowleri: contact-dependent secretion of electrondense granules (EDG).

The free living amoeba Naegleria fowleri is pathogenic to humans but also to other mammalians. These amoebae may invade the nasal mucosa and migrate into the brain causing cerebral hemorrhagic necrosis, a rapidly fatal infection. Knowledge of the cytolytic mechanism involved in the destruction of brain tissues by Naegleria trophozoites is limited. In other amoebic species, such as Entamoeba histolytica, we have previously reported the possible lytic role of small cytoplasmic components endowed with proteolytic activities, known as electrondense granules (EDG). Using transmission electron microscopy we now report that EDG, seldom found in long term cultured N. fowleri, are present in abundance in trophozoites recovered from experimental mice brain lesions. Numerous EDG were also observed in amoebae incubated with collagen substrates or cultured epithelial cells. SDS-PAGE assays of concentrated supernatants of these trophozoites, containing EDG, revealed proteolytic activities. These results suggest that EDG may have a clear role in the cytopathic mechanisms of this pathogenic amoeba.

Silicone hydrogel contact lenses surface promote Acanthamoeba castellanii trophozoites adherence: qualitative and quantitative analysis.

PURPOSE: To describe the adhesion properties of Acanthamoeba castellanii trophozoites to silicone hydrogel contact lenses of first generation (lotrafilcon A), second generation (galyfilcon A), and third generation (comfilcon A) and correlate the results with their specific surface characteristics, time of interaction, and suspension media. METHODS: Qualitative and quantitative assessments of the adhesion of 200 trophozoites of A. castellanii on contact lenses in culture medium (Bacto Casitone) and isotonic saline (IS) at different time points (15 minutes and 6 hours) were determined. RESULTS: By scanning electron microscopy, A. castellanii trophozoites were observed firmly adhered to the surface of hydrogel lenses after 15 minutes of interaction. The surface of lotrafilcon A lenses on which amoebae adhere better (16.4±10.2 amoebae/lens section) is rough and folded, which increases the contact surface with trophozoites, allowing acanthopodia to attach firmly. Contrarily, galyfilcon A lenses have a smoother surface, and lower numbers of amoebae were observed adhered to these lenses (4.7±2.9 amoebae/lens section). Even fewer amoebae adhered to the smoother surface of the comfilcon A lens (2.2±1.7 amoebae/lens section). Trophozoites showed similar behavior in both Bacto Casitone medium and IS. CONCLUSION: A rough surface may contribute to better adhesion of amoebae to silicone hydrogel lenses. Although a reduced numbers of trophozoites adhered to smooth lenses, trophozoites are a risk factor for amoebic keratitis. Isotonic saline facilitated trophozoite survival, suggesting that homemade saline solutions may contribute to the persistence of trophozoites, especially when there is no proper hygiene regimen used with the contact lens cases.

Acrylonitrile derivatives: In vitro activity and mechanism of cell death induction against Trypanosoma cruzi and Leishmania amazonensis.

Leishmaniasis and Chagas disease are parasitic infections that affect millions of people worldwide, producing thousands of deaths per year. The current treatments against these pathologies are not totally effective and produce some side effects in the patients. Acrylonitrile derivatives are a group of compounds that have shown activity against these two diseases. In this work, four novels synthetic acrylonitriles were evaluated against the intracellular form and extracellular forms of L. amazonensis and T. cruzi. The compounds 2 and 3 demonstrate to have good selectivity indexes against both parasites, specifically the compound 3 against the amastigote form (SI = 6 against L. amazonensis and SI = 7.4 against T. cruzi). In addition, the parasites treated with these two compounds demonstrate to produce a programmed cell death, since they were positive for the events studied related to this type of death, including chromatin condensation, accumulation of reactive oxygen species and alteration of the mitochondrial membrane potential. In conclusion, this work confirms that acrylonitriles is a source of possible new compounds against kinetoplastids, however, more studies are needed to corroborate this activity.

Correction: Castelan-Ramírez et al. Schwann Cell Autophagy and Necrosis as Mechanisms of Cell Death by Acanthamoeba. Pathogens 2020, 9, 458.

In the original publication [...].

Acanthamoeba royreba: morphological features and in vitro cytopathic effect.

Observations on cultured Acanthamoeba royreba trophozoites and in vitro cytopathogenicity of this amoeba are described. In culture, amoebae were active, pleomorphic and moved on the substrate by producing endocytic structures and emitting slight cytoplasmic microprojections from the cell surface. These projections were formed by hyaline cytoplasm and they were related to motion structures such as acanthopodia and lamellipodia, in which actin provides a framework that allows rapid changes in morphology. In the cytoplasm abundant vacuoles of different size and content were seen. By means of electron microscopy, it was possible to observe the compact fibrogranular appearance of the cytoplasm, along with the main cellular organelles such as the Golgi complex, the endoplasmic reticulum, digestive vacuoles, mitochondria and contractile vacuoles. Incubation of MDCK epithelial cell monolayers with conditioned medium did not produce a significant structural damage to the monolayer, even after 24h of incubation. When the trophozoites were incubated with the target cells the monolayer exhibited a clear injury created by the amoebae, which produced focal damage. Nevertheless, the rest of the monolayer appeared to remain intact, suggesting that a contact-dependent interaction is necessary to damage the target cells. These observations demonstrate the low invasive capacity of this amoeba.

Acanthamoeba castellanii cysts: new ultrastructural findings.

During Acanthamoeba castellanii trophozoite-cysts differentiation, four morphological stages were identified by scanning electron microscopy: trophozoite, precyst, immature cysts, and mature cysts. Fluorescence microscopy reveals the presence of small cumulus of actin in the cytoplasm of precysts after treatment with rhodamine phalloidin. By the contrary, in mature cysts, fluorescence was not observed. However, when excystation was induced, large fluorescent patches were present. By transmission electron microscopy, encysting amebas showed small cytoplasmic vesicles containing fibrillar material, surrounded by a narrow area of thin fibrils. Similar appearance was observed in pseudopods and phagocytic invaginations. In addition, large aggregates of rod-shape elements, similar to the chromatoid bodies, described in other amebas, were present in the cytoplasm. These cysts presented large areas with orange fluorescence after treatment with acridine orange.

Taurine, a Component of the Tear Film, Exacerbates the Pathogenic Mechanisms of Acanthamoeba castellanii in the Ex Vivo Amoebic Keratitis Model.

Acanthamoeba spp. is the etiological agent of amoebic keratitis. In this study, the effect of taurine in physiological concentrations in tears (195 µM) on trophozoites of Acanthamoeba castellanii through the ex vivo amoebic keratitis model was evaluated. Trophozoites were coincubated with the Syrian golden hamster cornea (Mesocricetus auratus) for 3 and 6 h. Group 1: Control (-). Corneas coincubated with amoebic culture medium and taurine. Group 2: Control (+). Corneas coincubated with trophozoites without taurine. Group 3: Corneas coincubated with taurine 15 min before adding trophozoites. Group 4: Trophozoites coincubated 15 min with taurine before placing them on the cornea. Group 5: Corneas coincubated for 15 min with trophozoites; subsequently, taurine was added. Results are similar for both times, as evaluated by scanning electron microscopy. As expected, in the corneas of Group 1, no alterations were observed in the corneal epithelium. In the corneas of Group 2, few adhered trophozoites were observed on the corneal surface initiating migrations through cell junctions as previously described; however, in corneas of Groups 3, 4 and 5, abundant trophozoites were observed, penetrating through different corneal cell areas, emitting food cups and destabilizing corneal surface in areas far from cell junctions. Significant differences were confirmed in trophozoites adherence coincubated with taurine (p < 0.05). Taurine does not prevent the adhesion and invasion of the amoebae, nor does it favor its detachment once these have adhered to the cornea, suggesting that taurine in the physiological concentrations found in tears stimulates pathogenic mechanisms of A. castellanii.

Acanthamoeba castellanii trophozoites that survive multipurpose solutions are able to adhere to cosmetic contact lenses, increasing the risk of infection.

Amoebae of the genus Acanthamoeba are etiological agents of amoebic keratitis, for which up to now there is no treatment of choice and one of its main risk factors is the use of contact lenses, including cosmetic contact lenses. Recently there has been an increase in amoebic keratitis cases due to the use of cosmetic contact lenses. Therefore, having a solution for the care of lenses with an efficient disinfectant effect that prevents the adhesion of trophozoites to lenses becomes essential. This study was carried out to determine the effect of 8 multipurpose contact lenses care solutions on Acanthamoeba castellanii trophozoites viability, and the efficiency of two of them to prevent the trophozoites adherence onto two cosmetic contact lenses (Acuvue 2, approved by the US Food and Drug Administration, and Magic Eye CCL, not approved). After 3 h of interaction, only AO Sept Plus, OPTI FREE Replenish, Renu Plus, Bio True and Multiplus significantly reduced the number of viable trophozoites with respect to the control; at 6 h Renu Plus, and at 12 h Conta Soft Plus and Multiplus, maintained the inhibitory effect. Only Opti Free Pure Moist did not significantly reduce the number of viable trophozoites. Multiplus and Opti Free Pure Moist (selected for their greater and lesser antiamibic effect) significantly reduced trophozoite adherence to both lenses; however, Opti Free Pure Moist was more efficient, despite the fact that A. castellanii adhered similarly to both lenses. Our results show that in all the multipurpose solutions evaluated, hundreds of viable A. castellanii trophozoites remain after several hours of incubation. Therefore, storage of the lenses in their case with MPS maintains the potential risk of amoebic keratitis in, cosmetic contact lenses wearers. Moreover, the use of CCL, not approved by the FDA, can increase the risk factor for AK since its poor manufacture can favor the permanence of amoebae, in addition to being a risk for corneal integrity.