Experimental keratitis in rats caused by Acanthamoeba griffini: A kinetic histopathological study.

The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with A griffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4 weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL-1ß, IL-10 and/or caspase 3. Intercellular adhesion molecule-1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4 weeks post-inoculation. By 8 weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by A griffini in a murine model.

Box Behnken design of siRNA-loaded liposomes for the treatment of a murine model of ocular keratitis caused by Acanthamoeba.

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.

Antimicrobial action of biguanides on the viability of Acanthamoeba cysts and assessment of cell toxicity.

PURPOSE: To assess dose- and concentration-dependent rates of biguanides on the viability of Acanthamoeba cysts isolated from severe ulcerative keratitis, and to correlate cysticidal activites with cytotoxic profiles in corneal and endothelial cells. METHODS: Cysticidal activities of polyhexamethylene biguanide and chlorhexidine digluconate were evaluated in the Acanthamoeba castellanii strain and clinical isolates of Acanthamoeba spp obtained from two severe and recurrent cases of ulcerative keratitis. The molecular characterization of protozoa used in the experimental assays was performed by sequencing reactions of the 18S rDNA gene. Acanthamoeba cysts were exposed at different dosages and concentrations of both biguanides; the application of double-biguanides was also evaluated. Automated cell viability assessment of cysts was performed using the trypan blue dye exclusion method. Cytotoxicity assays of biguanides were conducted using primary cultures of endothelial cells alone or in coculture with Acanthamoeba cysts. Human corneal epithelial cells were used as a comparative pattern to assess the toxicity of biguanide compounds. Cell viability was measured using both quantitative and qualitative methods. Statistical analyses were applied to the data. RESULTS: The in vitro study showed that all dosages, concentrations, and combinations of biguanides tested had a cysticidal effect on Acanthamoeba spp strains tested compared with control cultures not exposed to any antimicrobials; the difference in response was statistically significant. The use of both biguanides in combination demonstrated the best cysticidal effect. The use of isolated biguanides was associated with greater cytotoxic effects than with biguanides used in combination. Chlorhexidine digluconate used alone tended to have greater cytotoxicity than polyhexamethylene biguanide. Furthermore, the double-biguanide application had a statistically significant decrease in the deleterious effect on endothelial cells at higher dosage and concentration. Quantitative and qualitative analyses demonstrated the toxic effect of biguanide compounds on the viability of corneal epithelial cells, under single or in combination usage. CONCLUSIONS: We demonstrated that the combined use of biguanides had greater cysticidal activity than individual drug application as well as a possible protective effect on endothelial cells. The biguanide compounds tested were able to induce corneal epithelial cell death in time and concentration-independent fashions. Findings support the hypothesis concerning the cysticidal effect and the differential patterns of toxicity expressed by polyhexamethylene biguanide and chlorhexidine digluconate on the endothelial and corneal cells.

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.

Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis.

Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.

[Diagnosis of Acanthamoeba corneal infection by impression cytology: case report]. / Citologia de impressão no diagnóstico de infecção corneana por Acanthamoeba: relato de caso.

To describe three cases of corneal infection due to Acanthamoeba sp in which was possible to detect Acanthamoeba sp cysts by the corneal impression cytology technique. Three patients referred to the External Eye Disease Laboratory in 2004 with superficial corneal alterations were submitted to corneal specimen collection by impression cytology filter paper to investigate the presence of Acanthamoeba sp cysts. Two impression cytology samples were obtained from each patient and were stained by PAS, hematoxylin and Papanicolaou. Routine microbiological investigation and culture were also performed using corneal scraping. Positive culture and impression cytology for Acanthamoeba sp was observed in all patients while smears with Giemsa stain were positive in two. Impression cytology Acanthamoeba sp cysts were observed among sheets of corneal epithelial cells and as isolated cells. Cysts were also found in the superficial epithelium in one of these patients after treatment while corneal scraping did not reveal any cyst. Histopathology revealed cysts in the epithelium and stroma in a transplanted cornea in one of these patients. The first description of impression cytology as a diagnostic method for Acanthamoeba keratitis occurred recently. In this study corneal impression cytology detected Acanthamoeba sp cysts successfully in these patients with only superficial involvement. Impression cytology as a non invasive technique can be used to facilitate early recognition of Acanthamoeba infection playing a useful role in the follow-up of the disease.

Examen histopatológico a lente de contacto paciente con queratitis por Acanthamoeba: informe preliminar / Histopathologic examination to contact lens in patient with keratitis by Acanthamoeba: preliminary report

Un grupo de amebas de vida libre (AVL) es capaz de producir afecciones graves en la especie humana, tales como Meningoencefalitis Amebiana Primaria; Encefalitis Granulomatosa Amebiana; y Queratitis por colonización del epitelio corneal en individuos inmunocompetentes, especialmente en usuarios permanentes de lentes de contacto. Se procede a efectuar estudio histológico a lente de contacto proveniente de paciente de 45 años, con diagnóstico presuntivo de queratitis por Acanthamoeba sp. Y cuya muestra de raspado de córnea resulto positivo en medio de cultivo ANNE. Las muestras fueron teñidas con PAS y observadas al microscopio con 4x, 10x y 40x, observándose estructuras altamente sugerentes de quistes de este grupo de amebas. Por la importancia que requiere obtener un diagnóstico rápido y certero en esta patología ocular, debido a que los métodos tradicionales demoran alrededor de una semana, consideramos relevante dar a conocer este hallazgo citohistológico.

Induction of morphological and electrophysiological changes in hamster cornea after in vitro interaction with trophozoites of Acanthamoeba spp.

Acanthamoeba castellani and Acanthamoeba polyphaga are free-living amebae that cause keratitis and granulomatous encephalitis in humans. We have analyzed the early morphological and electrophysiological changes occurring during the in vitro interaction of cultured amebae with intact or physically damaged corneas obtained from hamsters. Both species of Acanthamoeba produced similar cytopathic changes, as seen by light microscopy and scanning electron microscopy. After adhesion to the epithelial surface, trophozoites formed clumps and migrated toward the cell borders, causing the separation of adjacent cells at 1 h of coculture. At later stages (2 to 4 h), some amebae were found under desquamating epithelial cells whereas others were seen associated with damaged cells or forming amebostome-like structures to ingest detached epithelial cells. Control corneas incubated in culture medium conditioned with amebae showed a cytoplasmic vacuolization and blurring of the epithelial-stromal junction. The early stages of corneal epithelial damage caused by amebae were also analyzed by measuring the transepithelial resistance changes in corneas mounted in Ussing chambers. Both species of Acanthamoeba caused a rapid decrease in electrical resistance. The present observations demonstrate that under in vitro conditions, Acanthamoeba trophozoites rapidly cause significant damage to the corneal epithelium. Furthermore, in our experimental model, previous physical damage to the corneas was not a prerequisite for the development of amebic corneal ulcerations.