Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis.

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.

A Rabbit Model of Acanthamoeba Keratitis: Use of Infected Soft Contact Lenses After Corneal Epithelium Debridement With a Diamond Burr.

Purpose: To develop a rabbit model of Acanthamoeba keratitis (AK) as the best method to reproduce the natural course of this disease. Methods: To induce AK, infected contact lenses (1000 amoebae/mm2, 90% trophozoites) were placed over the previously debrided corneal surface, in combination with a temporary tarsorrhaphy. Environmental and clinical strains of Acanthamoeba spp. (genotype T4) were used. Three groups (1L, n = 32; 2L-21d, n = 5; 2L-3d, n = 23) were established according to the number of contact lenses used (1L, 1 lens; 2L-21d and 2L-3d, 2 lenses) and the placement day of these (1L, day 1; 2L-21d, days 1 and 21; 2L-3d, days 1 and 3). The infection was quantified by a clinical score system and confirmed using corneal cytology and culture, polymerase chain reaction and histopathologic analysis. Results: The infection rate obtained was high (1L, 87.5%; 2L-21d, 100%; 2L-3d, 82.6%), although no clinical signs were observed in the 50% of the infected animals in group 1L. Among groups, group 2L-3d showed more cases of moderate and severe infection. Among strains, no statistically significant differences were found in the infection rate. In the control eyes, cross infection was confirmed when a sterile contact lens was placed in the previously debrided corneas but not if the eye remained intact. Conclusions: The combination of two infected contact lenses after corneal debridement seems to be an alternative model, clinically and histopathologically similar to its human counterpart, to induce the different AK stages and reproduce the course of the disease in rabbits.

Potential Value of Cellulose Synthesis Inhibitors Combined With PHMB in the Treatment of Acanthamoeba Keratitis.

PURPOSE: The aim of this study was to improve the cytopathic effect (CPE) of antiamebic agents by combining with cellulose synthesis inhibitor as an encystation inhibitor. METHODS: Cellulose synthesis inhibitors, 2,6-dichlorobenzonitrile (DCB) and isoxaben were used to block encystation of Acanthamoeba during cultivation. Cultured human corneal epithelial (HCE) cells and Acanthamoeba were treated with polyhexamethylene biguanide (PHMB) combined with cellulose synthesis inhibitors to evaluate the CPE as an antiamebic agent. RESULTS: 0.02% PHMB showed a 51.9% CPE on HCE cells within 30 minutes but exhibited significant toxic effects on Acanthamoeba. At a level of 0.00125%, PHMB had no significant CPEs on HCE cells, whereas 100 µM DCB and 10 µM isoxaben significantly inhibited the formation of the inner cyst wall of Acanthamoeba during encystation, and Acanthamoeba trophozoites failed to convert into mature cysts. Although a low concentration (0.00125%) of PHMB was used, the novel combinations with 100 µM DCB or 10 µM isoxaben had 23.4% or 18.7% additional amebicidal effects on Acanthamoeba. However, 100 µM DCB and 10 µM isoxaben had no CPEs on HCE cells. CONCLUSIONS: The combination of cellulose synthesis inhibitors with low concentrations of PHMB reduced the CPE on HCE cells and improved the amebicidal effect on Acanthamoeba by inhibition of encystation.

Reevaluating the role of Acanthamoeba proteases in tissue invasion: observation of cytopathogenic mechanisms on MDCK cell monolayers and hamster corneal cells.

The morphological analysis of the cytopathic effect on MDCK cell monolayers and hamster cornea and qualitative and quantitative analyses of conditioned medium and proteases were evaluated and compared between two strains of Acanthamoeba genotype T4. Further than highlighting the biological differences found between both strains, the most important observation in this study was the fact that proteases both in total extracts and in conditioned medium are apparently not determinant in tissue destruction. An interestingly finding was that no lysis of corneal tissue was observed as it was previously suggested. These results, together with previous studies, allow us to conclude that the invasion and disruption of corneal tissue is performed by the penetration of the amoebae through cell junctions, either by the action of proteases promoting cellular separation but not by their destruction and/or a mechanical effect exerted by amoebae. Therefore, contact-dependent mechanisms in Acanthamoeba pathogenesis are more relevant than it has been previously considered. This is supported because the phagocytosis of recently detached cells as well as those attached to the corneal epithelium leads to the modification of the cellular architecture facilitating the migration and destruction of deeper layers of the corneal epithelium.

Acanthamoeba-cytopathic protein induces apoptosis and proinflammatory cytokines in human corneal epithelial cells by cPLA2α activation.

PURPOSE: We have shown that Acanthamoeba interacts with a mannosylated protein on corneal epithelial cells and stimulates trophozoites to secrete a mannose-induced 133 kDa protease (MIP-133), which facilitates corneal invasion and induces apoptosis. The mechanism of MIP-133-induced apoptosis is unknown. The aim of this study was to determine if MIP-133 induces apoptosis and proinflammatory cytokines/chemokines in human corneal epithelial (HCE) cells via the cytosolic phospholipase A(2α) (cPLA(2α)) pathway. METHODS: HCE cells were incubated with or without MIP-133 at doses of 7.5, 15, and 50 µg/mL for 6, 12, and 24 hours. The effects of cPLA(2α) inhibitors on cPLA(2α), arachidonic acid (AA) release, and apoptosis were tested in vitro. Inhibition of cPLA(2α) involved preincubating HCE cells for 1 hour with cPLA(2α) inhibitors (10 µM methyl-arachidonyl fluorophosphonate [MAFP] or 20 µM arachidonyl trifluoromethyl ketone [AACOCF3]) with or without MIP-133 for 24 hours. Expression of cPLA(2α) mRNA and enzyme was examined by RT-PCR and cPLA(2) activity assays, respectively. Apoptosis of corneal epithelial cells was determined by caspase-3 and DNA fragmentation assays. Expression of IL-8, IL-6, IL-1ß, and IFN-γ was examined by RT-PCR and ELISA. RESULTS: MIP-133 induced significant cPLA(2α) (approximately two to four times) and AA release (approximately six times) from corneal cells while cPLA(2α) inhibitors significantly reduced cPLA(2α) (approximately two to four times) and AA release (approximately three times) (P < 0.05). cPLA(2α) inhibitors significantly inhibited MIP-133-induced DNA fragmentation approximately 7 to 12 times in HCE cells (P < 0.05). MIP-133 specifically activates cPLA(2α) enzyme activity in HCE cells, which is blocked by preincubation with anti-MIP-133 antibody. In addition, MIP-133 induced significant IL-8, IL-6, IL-1ß, and IFN-γ production, approximately two to three times (P < 0.05). CONCLUSIONS: MIP-133 interacts with phospholipids on plasma membrane of HCE cells and activates cPLA(2α). cPLA(2α) is involved in apoptosis, AA release, and activation of proinflammatory cytokines/chemokines from HCE cells. cPLA(2α) inhibitors may be a therapeutic target in Acanthamoeba keratitis.

Toll-like receptor 4 signalling pathway activation in a rat model of Acanthamoeba Keratitis.

The pathogenesis of Acanthamoeba keratitis (AK) is complicated. In our previous studies, TLR4 was found involved in the process of infection by Acanthamoeba in human corneal cells. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signalling pathway in Wistar rats challenged with Acanthamoeba. The rat model of AK was established. Corneas were collected and analysed by real-time PCR to assess the mRNA levels of TLR 2, 4, myeloid differentiation protein (MyD)88, nuclear factor (NF)-κB, extracellular signal-regulated kinase (ERK), interleukin (IL)-8, tumour necrosis factor (TNF)-α and interferon (IFN) -ß. Immunocytochemistry and Western blot were conducted to examine the proteins of TLR2, TLR4, p-Erk1/2 and p-IκB. Specific inhibitors PDTC and U0126 were used to pretreat the animals to determine the exact receptor and signalling pathway involved in pathogenesis. Expressions of TLR4, MyD88, all three cytokines, NF-κB, p-IκB and p-Erk1/2 were increased in Acanthamoeba-treated rat corneas. PDTC inhibited the production of IL-8 and TNF-α, while U0126 inhibited the synthesis of IFN-ß. TLR4 was involved in sensing the challenge of Acanthamoeba and inducing production of cytokines through TLR4-NF-κB and TLR4-Erk1/2 pathways in corneas of Wistar rats.

Acanthamoeba castellanii: morphological analysis of the interaction with human cornea.

The present study demonstrates that when Acanthamoeba castellanii trophozoites are co-cultivated with isolated human corneas, the amoeba can be invasive and cause damage to the intact corneal epithelium without the requirement of previous corneal abrasion. After adhesion, A. castellanii trophozoites migrate between cells forming bumps on the corneal cell layers and reaching Bowman s membrane in 3h, although no evidence of cell damage was observed until the phagocytic process was detected. Likewise, conditioned medium produced damage to the corneal cells that was proportional to the time of incubation, but this cytophatic effect involved only the most superficial layer of the human cornea and was not enough to explain amoebic invasion of Bowman s membrane. As a result of our observations, we suggest that the mechanical action of the trophozoites and phagocytosis of corneal cells during the process of corneal invasion are more important than previously suggested.

Effects of multipurpose contact lens care solutions on the adhesiveness of Acanthamoeba to corneal epithelial cells.

PURPOSE: To study the adhesion of Acanthamoeba castellanii treated with multipurpose contact lens care solutions (MPSs) to human corneal epithelial cells. METHODS: Cell suspensions of A. castellanii (ATCC50514) trophozoites were mixed with six MPSs: MPS-A (polyhexamethylene biguanide [PHMB], macrogolglycerol hydroxystearate, propylene glycol), MPS-B (PHMB, poloxamine, boric acid), MPS-C (polyquad, poloxamine, boric acid), MPS-D (PHMB, poloxamer, propylene glycol), MPS-E (PHMB, poloxamer), or MPS-F (PHMB, poloxamer) for 4 hr. Morphologic changes of A. castellanii after exposure with MPSs were observed with scanning electron microscopy. A. castellanii cells treated with MPS for 4 hr were inoculated onto cultured SV40-immortalized human corneal epithelial cells. After 2-hr incubation, the number of adherent A. castellanii was assessed by 18S-rDNA quantification using real-time polymer chain reaction. RESULTS: After 4-hr incubation, MPS-A- and MPS-B-treated A. castellanii have changed from trophozoite morphology into cyst form; however, MPS-E- and MPS-F-treated A. castellanii maintained trophozoite morphology. In contrast, both cyst and trophozoite forms were observed in MPS-C- and MPS-D-treated A. castellanii. The adherence rate of A. castellanii was approximately two times higher in MPS-E (not significant), and more than three times higher in MPS-F (P<0.05) compared with MPS-A, which produced the lowest adhesiveness as estimated by real-time polymer chain reaction. CONCLUSIONS: Taken together, these results support the possibility that chronic use of MPS with the lowest efficacies on promoting encystment of A. castellanii (MPS-E and MPS-F) by hydrogel contact lens wearers may increase adhesiveness of A. castellanii to corneal epithelial cells.

In vitro amoebacidal activity of photodynamic therapy on Acanthamoeba.

AIM: To evaluate the effectiveness of Hypocrellins B (HB) photodynamic therapy (PDT) on Acanthamoeba trophozoites and cysts, and its cytotoxic potential on the corneal epithelial cells and stromal cells in vitro. METHODS: Acanthamoeba trophozoites and cysts (5 x 10(4)/ml) were incubated with various concentrations of HB, followed by exposure to light (>470 nm) for 30 min at a fluence rate of 50 mW/cm(2). Survival rate was assessed using the live/dead viability/cytotoxicity assay and non-nutrient agar--Escherichia coli culture assay. Rabbit corneal epithelial cells and stromal cells were incubated with various concentrations of HB, followed by exposure to the same light fluence rate as above. The corneal cell survival rate was assessed using the live/dead viability/cytotoxicity assay. RESULTS: HB-PDT showed a dose-dependent inhibition on the trophozoites and cysts. The 100% inhibitory concentration (IC(100)) of HB was 1 microg/ml for the trophozoites and 20 microg/ml for the cysts. The 50% inhibitory concentration (IC(50)) of HB for the cysts was 3.8 microg/ml and for the trophozoites 0.23 microg/ml. The inhibition of the HB-PDT on the rabbit corneal cells and stromal cells was observed, but HB without irradiation had no inhibition on the corneal cells. CONCLUSION: HB-PDT had amoebacidal properties in vitro and showed a better effectiveness on the trophozoites than the cysts. Meanwhile, HB-PDT demonstrated cytotoxicity on the corneal epithelial cells and stromal cells, but HB exhibited no appreciable cytotoxicity in the dark to the corneal cells.

In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis.

OBJECTIVE: This study included in vivo and ex vivo investigations of patients with early-stage Acanthamoeba keratitis by using new-generation laser confocal microscopy (Heidelberg Retina Tomograph 2 Rostock Cornea Module [HRT 2-RCM]). METHODS: Three patients (2 men and 1 woman; mean age, 22.0 years) with early-stage Acanthamoeba keratitis diagnosed by direct examination (Parker ink-potassium hydroxide stain), culture from corneal epithelial scrapings, or both methods were enrolled in this study. All patients were examined by slit-lamp biomicroscopy. The area of the affected cornea was examined by HRT 2-RCM. Selected images of in vivo corneal layers and ex vivo cultured microorganisms were evaluated qualitatively for shape and degree of light reflection of the corneal structural changes or Acanthamoeba cysts. In addition, cultured Acanthamoeba were examined ex vivo by HRT 2-RCM. RESULTS: In vivo laser confocal microscopy showed highly reflective round-shaped, high-contrast Acanthamoeba cysts (10-20 microm in diameter) in the corneal epithelium in all cases, leading to rapid confirmation of the clinical diagnosis. In all culture samples of Acanthamoeba, ex vivo laser confocal microscopy showed highly reflective round- or stellate-shaped high-contrast particles (10-20 microm in diameter). CONCLUSIONS: In vivo laser confocal microscopy enables rapid and noninvasive diagnosis of early-stage Acanthamoeba keratitis with high resolution. In addition, ex vivo laser confocal images of Acanthamoeba cysts may be helpful when similar structures are identified and have to be interpreted under in vivo conditions.

Milk components inhibit Acanthamoeba-induced cytopathic effect.

PURPOSE: Acanthamoebae provoke a vision-threatening corneal infection known as Acanthamoeba keratitis (AK). It is thought that Acanthamoeba-specific IgA antibodies present in mucosal secretions such as human tears, milk, and saliva provide protection against infection by inhibiting the adhesion of parasites to host cells. The goal of the present study was to determine whether human mucosal secretions have the potential to provide protection against the Acanthamoeba-induced cytopathic effect (CPE) by an additional mechanism that is independent of IgA. METHODS: Breast milk was used as a model of human mucosal secretions. In vitro CPE assays were used to examine the CPE inhibitory effect of IgA-depleted milk and various milk fractions obtained by gel filtration. The activity of amebic proteinases was examined by zymography. RESULTS: IgA-depleted milk inhibited the Acanthamoeba-induced CPE in a concentration-dependent manner. Milk proteins were separated into four major fractions (F1-F4) by gel filtration. Of these four fractions, CPE inhibitory activity was detected largely in fraction F3. In contrast, fractions F1, F2, and F4 lacked CPE inhibitory activity. Moreover, fraction F3, but not F1, F2, or F4, inhibited amebic proteinases. CONCLUSIONS: These data, in conjunction with published findings showing that amebic proteinases are responsible for the induction of Acanthamoeba CPE, led us to propose that human mucosal secretions have the potential to provide protection against Acanthamoeba-induced CPE by an additional mechanism that is independent of IgA and that involves the inhibition of cytotoxic proteinases of amebae.

Effect of human tears on acanthamoeba-induced cytopathic effect.

OBJECTIVE: To determine whether tears of healthy individuals provide protection against Acanthamoeba-induced cytopathic effect (CPE) in vitro. METHODS: Acanthamoebae were added to confluent cultures of corneal epithelium in 24-well plates, and co-cultures were incubated overnight in a serum-free medium containing varying amounts of tears or immunoglobulin A (IgA)-depleted tears. At the end of the incubation period, the cells were stained with Giemsa, and the extent of target cell damage (ie, CPE) was quantified. RESULTS: Acanthamoebae produced extensive CPE. The presence of even a low concentration of tears (10 microL of undiluted tears per milliliter of media) almost completely inhibited Acanthamoeba-induced CPE. The CPE was inhibited by pretreatment of the parasites with tears. In contrast, the pretreatment of host cells with tears was not protective. This finding suggests that the target of the inhibitory factor is the parasite. IgA-depleted tears also inhibited Acanthamoeba-induced CPE, albeit with a lower potency than total tears. CONCLUSION: In addition to known IgA-dependent protective factors, human tears contain factors that inhibit Acanthamoeba-induced CPE independently of IgA. Clinical Relevance Identification and characterization of factors that protect against Acanthamoeba-induced CPE should help in the development of novel, rationally designed strategies to manage and protect against keratitis.

[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.

Acanthamoeba: keratopathogenicity of isolates from domestic tap water in Korea.

In a previous study, we reported on the contamination rate of free living amoeba, including Acanthamoeba, isolated from contact lens storage cases (CLSC) and domestic tap water in Korea. In an effort to evaluate the potential kerato-pathogenicity of 5 isolates from CLSC and 17 isolates from domestic tap water, we have conducted an investigation into the morphological features, mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) phenotypes, 18S rDNA sequences, and drug sensitivities of these isolates, and have compared the results with those of 20 amoebic keratitis (AK) isolates from Korea, as well as 14 reference strains. Cysts from 22 isolates obtained from CLSC and domestic tap water showed typical characteristics of morphological group 2. A total of three and five mtDNA RFLP patterns generated by EcoRI were found in 5 of the isolates from CLSC and 17 of the isolates from domestic tap water, respectively. The mtDNA RFLP patterns of four of the five isolates from the CLSC were found to be identical to those of the isolates from domestic tap water of students who had contaminated CLSC. The majority had mtDNA RFLP patterns identical to those of AK isolates in Korea. The results of 18S rDNA sequencing analysis were also shown to coincide with the results of mtDNA RFLP analysis. KA/WP12 was determined to be profoundly sensitive to chlorhexidine (MCC; 6.25microg/ml), and KAWP2 was the most sensitive strain to polyhexamethylene biguanide (PHMB) (MCC; 4.69microg/ml). Some difference in the cytopathic effects of isolates against human corneal epithelial cells was observed according to their mtDNA genotypes. In conclusion, domestic tap water may constitute a source of Acanthamoeba contamination of CLSC, and most isolates from CLSC and domestic tap water appear to be potentially keratopathogenic.

Intracorneal instillation of latex beads induces macrophage-dependent protection against Acanthamoeba keratitis.

PURPOSE: Instillation of sterile 1.0 microM latex beads into the central corneal epithelium renders Chinese hamsters resistant to corneal infection with Acanthamoeba castellanii. By contrast, activation of the adaptive immune response by subcutaneous immunization with A. castellanii antigens fails to protect against Acanthamoeba keratitis. This study was undertaken to examine the mechanisms that mediate latex bead-induced resistance to Acanthamoeba keratitis. METHODS: In vitro experiments examined the effect of latex bead treatment on the capacity of A. castellanii trophozoites to adhere to and kill corneal epithelial cells. In vivo administration of antineutrophil antiserum was used to evaluate the role of neutrophils in latex-bead-induced protection against Acanthamoeba keratitis. Liposomes containing the macrophagicidal drug clodronate were used to deplete conjunctival macrophages and determine the role of macrophages in the latex-bead-induced resistance. RESULTS: Latex bead treatment did not affect adherence of trophozoites to the corneal epithelium or protect corneal epithelial or stromal cells from trophozoite-mediated cytolysis in vitro. Neutrophil depletion did not abrogate the latex beads' protective effect. Latex bead treatment induced a significant infiltration of macrophages into the corneas that peaked at day 4 of infection. Moreover, depletion of conjunctival macrophages with the macrophagicidal drug clodronate eliminated the latex beads' protective effect. CONCLUSIONS: The results indicate that intracorneal injection of latex beads induces a remarkable resistance to Acanthamoeba keratitis that is largely, if not entirely, mediated by macrophages. These results underscore the importance of the innate immune apparatus in the resistance to Acanthamoeba keratitis.

Acanthamoeba keratitis associated with misuse of daily disposable contact lenses.

PURPOSE: To report a case of Acanthamoeba keratitis that occurred in a daily disposable contact lens wearer. METHOD: Case report. RESULTS: A 70-year-old gentleman presented to eye casualty with a red left eye with reduced vision and corneal epithelial changes. Acanthamoeba keratitis was not suspected initially as he was a daily disposable contact lens wearer. This led to a delay of 17 days in initiating treatment. He responded well to topical polyhexamethylene biguanide and propamidine and made a full recovery. CONCLUSION: Acanthamoeba keratitis can occur in patients who reuse daily disposable contact lenses. A diagnosis of Acanthamoeba keratitis can be difficult to make and should be considered in all patients who wear contact lenses, including daily disposable lenses. All patients who wear daily disposable contact lenses should be reminded that the benefits of this modality of contact lens are only possible if the lenses are worn once and thrown away.

In vitro pathogenicity of Acanthamoeba is associated with the expression of the mannose-binding protein.

PURPOSE: To determine whether the expression of Acanthamoeba mannose-binding protein (MBP) is associated with the pathogenicity of the parasite in vitro. METHODS: Both active trophozoites and dormant cysts of a pathogenic strain of A. castellanii were analyzed for their ability to bind to corneal epithelium, express MBP, and produce a cytopathic effect (CPE) on host cells. In addition, host cell binding, CPE-inducing ability, and MBP expression pattern of trophozoites of four different isolates of Acanthamoeba with various degrees of in vitro pathogenicity were analyzed. Binding assays were performed with radiolabeled parasites; CPE assays were performed with rabbit corneal epithelial cells as host cells; and the expression of MBP was detected by affinity chromatography of parasite extracts on mannose affinity columns and by immunohistochemical and Western blot analyses. RESULTS: Trophozoites of A. castellanii bound avidly to corneal epithelial cells in a mannose-inhibitable manner, whereas cysts exhibited little binding. The lack of binding of the cysts to host cells was associated with the downregulation of MBP, along with the concomitant loss of CPE. Analysis of trophozoites of five different species of Acanthamoeba exhibiting various degrees of pathogenic potential revealed that the ability of parasites to bind to host cells and produce CPE is directly correlated with the expression of the MBP. Acanthamoeba strains that bound avidly to host cells and produced potent CPE, robustly expressed MBP. In contrast, parasite strains that produced only weak CPE, expressed markedly reduced levels of MBP. CONCLUSIONS: The data demonstrating that the pathogenic potential of Acanthamoeba directly correlates with the expression level of the MBP in conjunction with our published studies showing that Acanthamoeba MBP is a major virulence protein suggest that the amoeba lectin has the potential to serve as a marker of pathogenicity.

Heidelberg retina tomograph II findings of Acanthamoeba keratitis.

PURPOSE: Heidelberg retina tomograph II (HRTII) examination was performed with cornea module in one patient with Acanthamoeba keratitis (AK) to provide images detailing characteristic findings of the disease. METHODS: A 34-year-old woman presented with clinical signs and symptoms of AK. HRTII with cornea module was performed and the patient underwent laboratory investigations. RESULTS: HRTII examination with cornea module revealed numerous 20-26-micro m diameter high-contrast round particles within the corneal epithelium and anterior stroma, resembling Acanthamoeba cysts. Stellate cells as well as ovoid irregular objects, possibly inflammatory cells, trophozoites, altered cysts, or activated keratocytes, were also present in the area of stromal infiltrates. Laboratory investigations confirmed the diagnosis of AK. CONCLUSIONS: HRTII cornea module can be helpful in the diagnosis of AK by identifying acanthamoeba cyst-like structures in the cornea. This technique also has potential uses in monitoring the efficiency of anti-infective treatment.