Differentiation of Acanthamoeba strains from infected corneas and the environment by using restriction endonuclease digestion of whole-cell DNA.
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Restriction endonuclease digestion of Acanthamoeba whole-cell DNA was used to study the relationship between 33 morphologically identical strains from keratitis cases (30 strains), contact lens storage containers (2 strains), and soil (1 strain). Samples digested with BglII, EcoRI, or HindIII and separated by agarose gel electrophoresis contained detectable mitochondrial DNA restriction fragment length polymorphisms (RFLPs). By comparing RFLPs, the strains could be assigned to seven multiple-strain and three single-strain groups. The largest of these contained nine strains, eight of which were isolated in keratitis cases in various locations worldwide and may indicate a group particularly associated with keratitis. Restriction endonuclease analysis of whole-cell DNA is proposed as a valuable technique for detecting mitochondrial DNA RFLPs in the differentiation of morphologically identical Acanthamoeba strains and may therefore be useful in resolving the complex taxonomy of the genus, which has hitherto been founded on subjective morphological criteria. (+info)
Microbiological diagnosis of infective keratitis: comparative evaluation of direct microscopy and culture results.
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AIMS: To determine the sensitivity, specificity and predictive values of potassium hydroxide (KOH) wet mount, Gram stain, Giemsa stain and Kinyoun's acid-fast stain in the diagnosis of infective keratitis. METHODS: A retrospective analysis of all patients with clinically diagnosed infective keratitis presenting between September 1999 and September 2002 was carried out. Corneal scrapes were taken and subjected to direct microscopy and culture. RESULTS: 3298 eyes of 3295 consecutive patients with infective keratitis were evaluated, of which 1138 (34.51%) eyes had fungal growth alone, 1069 (32.41%) had bacterial growth alone, 33 (1%) had Acanthamoeba growth alone, 83 (2.5%) had mixed microbial growth and the remaining 975 (29.56%) had no growth. The sensitivity of KOH wet mount was higher (99.3%; 95% confidence interval (CI) 98.6 to 99.6) in the detection of fungi, 100% (95% CI 90.4 to 100) in the detection of Nocardia and 91.4% (95% CI 75.8 to 97) in the detection of Acanthamoeba) than that of Gram-stained smears (89.2% (95% CI 87.3 to 90.8) in fungi, 87% (95% CI 73.0 to 94.6) in Nocardia and 60% (95% CI 42.2 to 75.6) in the detection of Acanthamoeba) in the detection of fungi, Nocardia and Acanthamoeba. 1764 of 3295 (53.54%) patients presented more than 7 days after onset of illness and 84.69% of the eyes had corneal ulcers with size >2 mm in diameter. Positivities of KOH (44.46%; p<0.001) and Gram-stained smears (77.37%; p<0.001) were found to be higher among eyes with larger ulcers (>2 mm) than among eyes with smaller ulcers (<2 mm). CONCLUSION: KOH smear is of greater diagnostic value in the management of infective keratitis, and it is recommended in all clinics without exception for establishing timely treatment. (+info)
Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis.
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BACKGROUND: The early microbiological diagnosis of corneal infections may prevent the condition from worsening. AIM: To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. METHODS: Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. RESULTS: The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). CONCLUSIONS: The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis. (+info)
Oral immunization with Acanthamoeba castellanii mannose-binding protein ameliorates amoebic keratitis.
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Acanthamoeba castellanii mannose-binding protein (MBP) mediates adhesion of the amoebae to corneal epithelial cells, a key first step in the pathogenesis of Acanthamoeba keratitis (AK), a devastating corneal infection. In the present study, we demonstrate that oral immunization with recombinant MBP ameliorates AK in a hamster animal model and that this protection is associated with the presence of elevated levels of anti-MBP immunoglobulin A in the tear fluid of the immunized animals. (+info)
Intracorneal instillation of latex beads induces macrophage-dependent protection against Acanthamoeba keratitis.
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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. (+info)
Effect of caspofungin on trophozoites and cysts of three species of Acanthamoeba.
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OBJECTIVES: Amoebic keratitis is difficult to treat, without total efficacy in some patients because of cysts that are less susceptible than trophozoites to the usual treatments. We investigated here the in vitro effectiveness of caspofungin, a new antifungal, against three species of Acanthamoeba. METHODS: Trophozoites and cysts of Acanthamoeba castellanii, Acanthamoeba culbertsoni and Acanthamoeba polyphaga were incubated with caspofungin at concentrations varying from 16 to 500 mg/L. RESULTS: The trophozoites of the three tested species were susceptible in vitro to caspofungin at a concentration of 250 mg/L (206 microM). Furthermore, this drug was cysticidal at a concentration of 500 mg/L (412 microM) against A. castellanii and A. culbertsoni. CONCLUSIONS: Caspofungin could represent, if in vivo studies confirm its efficacy, a new anti-Acanthamoeba compound. (+info)
Molecular characterization of Acanthamoeba isolated from amebic keratitis related to orthokeratology lens overnight wear.
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In an effort to characterize, on the molecular scale, the Acanthamoeba initially isolated from the cornea of an amoebic keratitis patient associated with overnight-wear orthokeratology lens in Korea, we conducted mitochondrial DNA restriction fragment length polymorphism, 18S rDNA sequencing, and drug sensitivity analyses on the isolate (KA/PE1). The patient was treated with polyhexamethylene biguanide, chlorhexidine and oral itraconazole, which resulted in resolution of the patientos ocular inflammation. The majority of the molecular characteristics of the KA/PE1 were determined to be identical, or quite similar, to those of A. castellanii Ma strain, which had been isolated also from amoebic keratitis. The risk of Acanthamoeba keratitis as a potential complication of overnight orthokeratology is briefly discussed. (+info)
Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence.
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The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba. (+info)