Simon Kilvington

Acanthamoeba keratitis: diagnosis and treatment update 2009.

PURPOSE To describe the current management of Acanthamoeba keratitis (AK). DESIGN A perspective based on the literature and author experience. RESULTS Early diagnosis and appropriate therapy are key to a good prognosis. A provisional diagnosis of AK can be made using the clinical features and confocal microscopy, although a definitive diagnosis requires culture, histology, or identification of Acanthamoeba deoxyribonucleic acid by polymerase chain reaction. Routine use of tissue diagnosis is recommended, particularly for patients unresponsive to treatment for AK. Topical biguanides are the only effective therapy for the resistant encysted form of the organism in vitro, if not always in vivo. None of the other drugs that have been used meet the requirements of consistent cysticidal activity and may have no therapeutic role. The use of topical steroids is controversial, but probably beneficial, for the management of severe corneal inflammatory complications that have not responded to topical biguanides alone. The scleritis associated with AK is rarely associated with extracorneal invasion and usually responds to systemic anti-inflammatory treatment combined with topical biguanides. Therapeutic keratoplasty retains a role for therapy of some severe complications of AK but not for initial treatment. With modern management, 90% of patients can expect to retain visual acuity of 6/12 or better and fewer than 2% become blind, although treatment may take 6 months or more. CONCLUSIONS Better understanding of the pathogenesis of the extracorneal complications, the availability of polymerase chain reaction for tissue diagnosis, and effective licensed topical anti-amoebics would substantially benefit patients with AK.

Comparison of Hydrogen Peroxide Contact Lens Disinfection Systems and Solutions against Acanthamoeba polyphaga

ABSTRACT Acanthamoeba is a free-living amoeba causing a potentially blinding infection of the cornea. Contact lens wearers are most at risk and account for some 95% of cases. Hydrogen peroxide is used for contact lens disinfection due to its broad antimicrobial activity. Lenses must be neutralized before use to avoid pronounced stinging and possible corneal damage. Neutralization is achieved by adding a catalyst during the disinfection process (one-step) or afterwards (two-step). Here, the activities of commercial peroxide systems and individual solutions against trophozoites and cysts ofAcanthamoeba polyphaga were compared. All disinfection systems were active against trophozoites, giving a ≥3-log (99.9%) kill within 1 h. Of the four one-step systems, only one showed some cysticidal activity, giving a 1.28 ± 0.41-log reduction. Both two-step systems were cysticidal, giving a ≥3-log kill at 4 h. All system peroxide solutions were cysticidal, giving a ≥3-log kill by 4 to 6 h. Variation in the cysticidal rate was observed with two solutions that gave a 1.8- to 2.1-log kill at 4 h compared with 3.0 to 4.0 for the rest (P < 0.05). No cysticidal activity was found with the peroxigen sodium perborate or the contact lens protein remover subtilisin A. Two-step systems are cysticidal providing contact times of at least 4 h are employed. Variation in cyst killing occurs between peroxide solutions, possibly due to formulation differences. One-step systems are less effective againstAcanthamoeba cysts due to rapid peroxide neutralization. The cysticidal activity of one-step systems could be improved if neutralization rates were retarded.

Persistently culture positive acanthamoeba keratitis: In vivo resistance and in vitro sensitivity

PURPOSE To characterize the risk factors, clinical course, treatment outcome and the association between in vivo resistance and in vitro sensitivity for subjects with persistently culture-positive Acanthamoeba keratitis. DESIGN Retrospective noncomparative case series. PARTICIPANTS Eleven subjects with repeatedly positive cultures for Acanthamoeba treated between January 1990 and December 2000, were reviewed. Only subjects with 2 or more positive cultures, availability of the clinical data, and availability of the last Acanthamoeba isolate were included in this study. METHODS The medical records were analyzed, and the last isolate from each case was tested in vitro for the antiamoebic drugs used clinically: polyhexamethylene biguanide (PHMB), chlorhexidine, propamidine and hexamidine. MAIN OUTCOME MEASURES Risk factors, the clinical outcome and in vitro cysticidal drug sensitivity assay. RESULTS Eleven subjects (11/180, 6.1%) had 2 or more positive cultures of whom 8 eyes of 8 subjects (8/180, 4.45%) were included in this study. Seven of eight (87%) subjects were diagnosed over 1 month from onset (late diagnosis). The most common presenting findings were diffuse stromal infiltrate (5/8, 62.5%), ring infiltrate (5/8, 62.5%), and corneal ulceration (3/8, 37.5%). The clinical course of the disease in all subjects consisted of recurrent episodes of corneal and scleral inflammation, with a mean duration of 13.4 +/- 9 months. All subjects received PHMB, and 5/8 (62.5%) chlorhexidine too; hexamidine was used in combination in 6/8 (75%), and propamidine in 1/8 (12.5%). All subjects had topical steroids, and 5/8 (62.5%) systemic immunosuppression. The disease resolved with corneal scarring in 3/8 (37.5%) subjects, corneal (or impending) perforation treated with therapeutic keratoplasty in 4/8 (50%), and enucleation in 1/8 (12.5%). Final visual acuity was 0.43 +/- 0.37. In vitro most isolates were resistant to propamidine, hexamidine was cysticidal in high concentrations, and PHMB and chlorhexidine had excellent sensitivity profiles. CONCLUSIONS In our large series of Acanthamoeba keratitis with a positive microbiologic diagnosis at presentation, nearly 5% developed recurrent episodes of corneal and scleral inflammation with viable Acanthamoeba in the cornea despite prolonged treatment with biguanides and/or diamidines. There was no correlation between in vitro drug sensitivities and the in vivo response for biguanides.

Solar disinfection of poliovirus and Acanthamoeba polyphaga cysts in water – a laboratory study using simulated sunlight

Aims:  To determine the efficacy of solar disinfection (SODIS) in disinfecting water contaminated with poliovirus and Acanthamoeba polyphaga cysts.

Encystment of Acanthamoeba during incubation in multipurpose contact lens disinfectant solutions and experimental formulations.

Objectives. Acanthamoeba keratitis (AK) is most commonly found in contact lens wearers with poor lens care hygiene. Recent reports have indicated an increase in cases of AK in the United States associated with the use of Complete MoisturePlus multipurpose solution. This association may not appear explainable by differences in Acanthamoeba kill rates among multipurpose solutions. In this study the physiologic response of Acanthamoeba trophozoites to incubation in various contact lens solutions was investigated. Methods. Acanthamoeba trophozoites were incubated in commercial and experimental solutions and the percentage encystment and viability determined in conjunction with microscopic analysis of cyst formation. Results. Among commercial products, MoisturePlus solution induced encystment within 6 to 24 hr of incubation. Only immature cysts formed which failed to mature even after 5 days incubation in the solution although they remained viable. Experimental formulations of MoisturePlus solution, modified by omitting key ingredients, identified propylene glycol as the agent responsible for inducing encystment. However, tests of other commercial solutions containing propylene glycol produced reduced or no encystment. Conclusions. The presence of propylene glycol was shown to be the key factor in inducing Acanthamoeba encystment. However, the reduced or absence of encystment in other commercial solutions containing propylene glycol suggests additional factors, such the buffering system, may be involved in the phenomenon. How these observations relate to the solution’s role in the reported association with AK is unclear. Nevertheless, it is recommended that all lens care products be tested for propensity to induce Acanthamoeba encystment and formulations containing propylene glycol may warrant special scrutiny.

Acanthamoeba polyphaga Strain Age and Method of Cyst Production Influence the Observed Efficacy of Therapeutic Agents and Contact Lens Disinfectants

ABSTRACT The effects of age in culture and the type of medium used for induction of Acanthamoeba polyphaga (Ros) cysts on susceptibilities to polyhexamethylene biguanide (PHMB; 3 μg/ml), chlorhexidine digluconate (30 μg/ml), myristamidopropyl dimethylamine (20 μg/ml), H2O2 (3%), and two multipurpose contact lens solutions (MPS-1 and MPS-2, based on 1 μg of PHMB per ml) were examined. Strain Ros-02 was cryopreserved on isolation in 1991, while strain Ros-91 had been in continuous axenic culture. Significant differences in susceptibilities to the disinfectants were found depending on the medium used for cyst preparation and the age of the test strain, with Ros-02 generally being more resistant. For example, the killing of Ros-91 cysts produced from an axenic culture of trophozoites in the presence of 50 mM MgCl2 by MPS-2 was 4 logs, but the killing of Ros-02 by MPS-2 was only 2 logs (P < 0.05) and killing of both strains with cysts obtained from monoxenic cultures with Escherichia coli was only 1 log (P < 0.001). Assays repeated with different batches of the various cyst types gave consistent results. A batch of Ros-91 cysts stored at 4°C and tested over an 8-week period with MPS-1 showed progressively increasing susceptibility to disinfection, although there was no loss of viability during storage (P < 0.01). These observations have important implications for the standardization and interpretation of Acanthamoeba disinfectant and therapeutic agent testing.

Activities of Therapeutic Agents and Myristamidopropyl Dimethylamine against Acanthamoeba Isolates

ABSTRACT The activities of therapeutic agents and myristamidopropyl dimethylamine (MAPD) against Acanthamoeba strains recalcitrant to medical therapy were studied. MAPD minimum cysticidal concentrations were 6.25 to 25 μg/ml; 10 to 30 μg/ml gave at least a 3-log cyst kill after 6 h, and 50 and 100 μg/ml gave at least a 3-log cyst kill within 2 and 1 h, respectively.

Identification and susceptibility to multipurpose disinfectant solutions of bacteria isolated from contact lens storage cases of patients with corneal infiltrative events

Corneal infiltrative events (CIEs) are being reported with increasing frequency in lens wearers and may be related to specific multipurpose disinfecting solution (MPDS), contact lens type or bacterial bio-burden. Here, the efficacy of MPDSs against bacteria from contact lens storage cases (CLSC) of patients with CIEs was investigated. Eighteen CLSC from patients with CIEs were cultured. All reported using the same MPDS based on PQ-1+Aldox+nonanoyl-EDTA prior to experiencing CIEs. Bacteria were identified and tested for sensitivity to MPDS-1 and three other MPSDs. 16/18 CLSC (89%) contained bacterial counts of ≥10(4)-10(8)/mL. Achromobacter spp. was most frequently identified and was found in 11/18 cases (61%). This was followed by 4/18 (22%) Stenotrophomonas maltophilia, 3/18 (17%) Serratia marcescens, 3/18 (17%) Delftia spp., 2/18 (11%) Elizabethkingia spp., 2/18 (11%) Chryseobacterium indologenes and 1/18 Sphingobacterium spiritivorum. Acanthamoeba was not isolated. All of the Achromobacter strains were resistant to MPDS-1 with <1log10 kill up to 14 days exposure and the solution also showed reduced efficacy against the other isolates at the manufacturers recommended disinfection time of 6h. Two strains of S. maltophilia and Delftia spp. grew in the solution over 14 days. Factors responsible for causing adverse events such as CIEs in contact lens wearers remain unclear. However, the presence of significant bio-burden in the contact lens storage case and lens may initiate an immunological response resulting in CIEs either directly or through the release of endotoxins (e.g. lipopolysaccharides) from the bacterial outer cell membrane.

The efficacy of simulated solar disinfection (SODIS) against Ascaris, Giardia, Acanthamoeba, Naegleria, Entamoeba and Cryptosporidium

The antimicrobial activity of simulated solar disinfection (SODIS) in the presence and absence of riboflavin against various protozoa and helminth organisms was investigated in this study. Assays were conducted in transparent 12 well microtitre plates containing a suspension of test organisms in the presence or absence of 250 μM riboflavin. Plates were exposed to simulated sunlight at an optical irradiance of 550 Wm(-2) (watts per square metre) delivered from a SUNTEST™ CPS+ solar simulator. Aliquots of the test suspensions were taken at set time points and the viability of the test organisms was determined by either culture, microscopy or flow cytometry where applicable. With Acanthamoeba, Naegleria, Entamoeba and Giardia exposure to SODIS at an optical irradiance of 550 Wm(-2) for up to 6h resulted in significant inactivation of these organisms. The addition of riboflavin to this system significantly increased the level of inactivation observed with cysts of A. castellanii. With Cryptosporidium oocysts and Ascaris ova exposure to SODIS in the presence and absence of riboflavin for 6-8h resulted in a negligible reduction in viability of both organisms. In this present study we have been able to show that SODIS is effective against a variety of previously untested waterborne organisms and with A. castellanii cysts the addition of micro-molar concentrations of riboflavin can enhance cyst inactivation. However, care must be taken as Ascaris larvae continue to develop inside the ova after exposure to SODIS and Cryptosporidium remain impermeable to propidium iodide staining indicating they may still be infectious.

Acidified nitrite enhances hydrogen peroxide disinfection of Acanthamoeba, bacteria and fungi

OBJECTIVES In the human innate immune system, stimulated phagocytes release reactive nitrogen intermediates that can react with superoxide to form the powerful oxidant peroxynitrite and other less abundant species. In this study, the efficacy of hydrogen peroxide (H2O2) and acidified nitrite (NaNO2) alone and in combination was compared against a variety of bacteria, fungi and protozoa. METHODS Challenge test assays based on the international standard (ISO 14729) were used to determine the antimicrobial activity of H2O2 and acidified NaNO2 at pH 5 alone and in combination against Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Mycobacterium aurum, Bacillus subtilis spores, Candida albicans, Fusarium solani conidia and Acanthamoeba polyphaga trophozoites and cysts. RESULTS When tested alone, both H2O2 (0.4% v/v) and NaNO2 (2 mg/mL, pH 5) produced a >or=4 log reduction in viability after 4 h of exposure for all bacteria and A. polyphaga trophozoites, but not B. subtilis spores, F. solani and A. polyphaga cysts, which gave a <or=1 log kill after 6 h, and M. aurum, which gave a <1 log kill with NaNO2 only. However, the combination of H2O2 and NaNO2 resulted in a >or=4 log kill of all test organisms within 1 h. Addition of NaNO2 also enhanced the antimicrobial efficacy of a H2O2-based contact lens disinfection system. CONCLUSIONS The findings of this study demonstrate that acidified NaNO2 can significantly enhance the antimicrobial activity of H2O2 probably through the generation of peroxynitrite. The addition of acidified nitrite to 3% (v/v) H2O2 solution may represent an improved one-step method for the disinfection of contact lenses, especially against highly resistant cysts of Acanthamoeba spp.