Prevalence and seasonal variation of Acanthamoeba in domestic tap water in greater Sydney, Australia.

BACKGROUND: This study examined the prevalence of free-living Acanthamoeba in domestic tap water in the greater Sydney region, Australia, and determined any seasonal variation in prevalence. METHODS: Fifty-four participants were included in this study following approval from an institutional human research ethics committee. Each participant self-collected two samples (one in summer and another in winter) from the surface of the drain of the bathroom sink using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli and incubation at 32°C for two weeks. The plates were microscopically examined for the presence of free-living amoeba. DNA was isolated from 20 samples and a polymerase chain reaction (PCR) assay was performed for amplification of the partial sequence of the 18S ribosomal RNA gene. The PCR amplified products were sequenced using Sanger sequencing and genotyping was performed based on the variation in nucleotide sequences. RESULTS: A total of 97 samples were collected over the two collection periods, with 28.6 per cent of samples morphologically classified as Acanthamoeba. The summer period yielded 16 of 54 (29.6 per cent) samples classified as Acanthamoeba, while the winter period yielded 12 of 43 (27.9 per cent) samples classified as Acanthamoeba. There was no statistically significant difference (p = 0.85) between the prevalence of free-living Acanthamoeba in summer compared to winter. Phylogenetic analysis showed that 15 of 20 (75 per cent) isolates belonged to genotype T4, the most frequent genotype isolated in Acanthamoeba keratitis. CONCLUSION: The prevalence of free-living Acanthamoeba characterised morphologically in domestic tap water of the greater Sydney region was higher than expected, especially considering the low incidence of Acanthamoeba keratitis in Australia. However, this study did not find variation between seasons. As the T4 genotype was most common, Sydney-based practitioners must always consider Acanthamoeba as a possible causative organism in cases of microbial keratitis, regardless of the season.

The relationship between environmental sources and the susceptibility of Acanthamoeba keratitis in the United Kingdom.

PURPOSE: To determine whether Acanthamoeba keratitis (AK) patients have higher rates of Acanthamoeba and free-living amoeba (FLA) colonising domestic sinks than control contact lens (CL) wearers, and whether these isolates are genetically similar to the corneal isolates from their CL associated AK. METHODS: 129 AK patients from Moorefield Eye Hospital, London and 64 control CL wearers from the Institute of Optometry were included in this study. The participants self-collected home kitchen and bathroom samples from tap-spouts, overflows and drains using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli, incubated at 32°C and examined for amoebae by microscopy for up to 2 weeks. Partial sequences of mitochondrial cytochrome oxidase genes (coxA) of Acanthamoeba isolates from four AK patients were compared to Acanthamoeba isolated from the patient's home. The association between sampling sites was analysed with the chi-square test. RESULTS: A total of 513 samples from AK patients and 189 from CL controls were collected. The yield of FLA was significantly greater in patients' bathrooms (72.1%) than CL controls' bathrooms (53.4%) (p<0.05). Spouts (kitchen 6.7%, bathroom 11%) had the lowest rate of Acanthamoeba isolation compared to drains (kitchen 18.2%, bathroom 27.9%) and overflow (kitchen 39.1%, bathroom 25.9%) either in kitchens or bathrooms (p<0.05). There was no statistically significant difference between the average prevalence of Acanthamoeba in all three sample sites in kitchens (16.9%) compared to all three sample sites in bathrooms (21.5%) and no association for Acanthamoeba prevalence between AK patients and CL controls. All four corneal isolates had the same coxA sequence as at least one domestic water isolate from the patients' sink of the kitchen and the bathroom. CONCLUSION: The prevalence of Acanthamoeba and FLA was high in UK homes. FLA colonisation was higher in AK patients compared to controls but the prevalence of Acanthamoeba between AK patients and CL controls domestic sinks was similar. This study confirms that domestic water isolates are probably the source of AK infection. Advice about avoiding water contact when using CL's should be mandatory.

Antimicrobial efficacy of a novel povidone iodine contact lens disinfection system.

PURPOSE: Contact lens (CL) wear is a risk factor for the acquisition of microbial keratitis. Accordingly, compliance to manufacturers' recommended hygiene and disinfection procedures are vital to safe (CL) use. In this study we evaluated a novel povidone-iodine (PI) (CL) disinfection system (cleadew, Ophtecs Corporation, Japan) against a range of bacterial, fungal and Acanthamoeba. METHODS: Antimicrobial assays were conducted according to ISO 14729 using the recommended strains of bacteria and fungi, with and without the presence of organic soil. Regrowth of bacteria and fungi in the disinfection system was also examined. The activity on biofilms formed from Stenotrophomonas maltophilia and Achromobacter sp. was evaluated. Efficacy against A. castellanii trophozoites and cysts was also investigated. RESULTS: The PI system gave >4 log10 kill of all bacteria and fungi following the manufacturer's recommended disinfection and cleaning time of 4h, with or without the presence of organic soil. No regrowth of organisms was found after 14days in the neutralized solution. In the biofilm studies the system resulted in at least a 7 log10 reduction in viability of bacteria. For Acanthamoeba, >3 log10 kill of trophozoites and 1.1-2.8 log10 kill for the cyst stage was obtained. CONCLUSIONS: The PI system effective against a variety of pathogenic microorganisms under a range of test conditions. Strict compliance to recommended CL hygiene procedures is essential for safe CL wear. The use of care systems such as PI, with broad spectrum antimicrobial activity, may aid in the prevention of potentially sight threatening microbial keratitis.

Fibrous Catalyst-Enhanced Acanthamoeba Disinfection by Hydrogen Peroxide.

SIGNIFICANCE: Hydrogen peroxide (H2O2) disinfection systems are contact-lens-patient problem solvers. The current one-step, criterion-standard version has been widely used since the mid-1980s, without any significant improvement. This work identifies a potential next-generation, one-step H2O2, not based on the solution formulation but rather on a case-based peroxide catalyst. PURPOSE: One-step H2O2 systems are widely used for contact lens disinfection. However, antimicrobial efficacy can be limited because of the rapid neutralization of the peroxide from the catalytic component of the systems. We studied whether the addition of an iron-containing catalyst bound to a nonfunctional propylene:polyacryonitrile fabric matrix could enhance the antimicrobial efficacy of these one-step H2O2 systems. METHODS: Bausch + Lomb PeroxiClear and AOSept Plus (both based on 3% H2O2 with a platinum-neutralizing disc) were the test systems. These were tested with and without the presence of the catalyst fabric using Acanthamoeba cysts as the challenge organism. After 6 hours' disinfection, the number of viable cysts was determined. In other studies, the experiments were also conducted with biofilm formed by Stenotrophomonas maltophilia and Elizabethkingia meningoseptica bacteria. RESULTS: Both control systems gave approximately 1-log10 kill of Acanthamoeba cysts compared with 3.0-log10 kill in the presence of the catalyst (P < .001). In the biofilm studies, no viable bacteria were recovered following disinfection in the presence of the catalyst compared with ≥3.0-log10 kill when it was omitted. In 30 rounds' recurrent usage, the experiments, in which the AOSept Plus system was subjected to 30 rounds of H2O2 neutralization with or without the presence of catalytic fabric, showed no loss in enhanced biocidal efficacy of the material. The catalytic fabric was also shown to not retard or increase the rate of H2O2 neutralization. CONCLUSIONS: We have demonstrated the catalyst significantly increases the efficacy of one-step H2O2 disinfection systems using highly resistant Acanthamoeba cysts and bacterial biofilm. Incorporating the catalyst into the design of these one-step H2O2 disinfection systems could improve the antimicrobial efficacy and provide a greater margin of safety for contact lens users.

The Characterization of an Adrenergic Signalling System Involved in the Encystment of the Ocular Pathogen Acanthamoeba spp.

The aim of this study was to identify and characterize the receptor system involved in controlling encystment in Acanthamoeba using specific agonists and antagonists and to examine whether endogenous stores of catecholamines are produced by the organism. Acanthamoeba trophozoites suspended in axenic growth medium were exposed to adrenoceptor agonists and antagonists to determine which compounds promoted or prevented encystment. Second, trophozoites were cultured in medium containing a catecholamine synthesis inhibitor to investigate the effect this had on natural encystment. Nonspecific adrenoceptor agonists including epinephrine, isoprotenerol, and the selective ß1 adrenoceptor agonist dobutamine were found to cause > 90% encystment of Acanthamoeba trophozoites compared to < 30% with the controls. The selective ß1 antagonist metoprolol was able to inhibit epinephrine mediated encystment by > 55%. Cultures of Acanthamoeba with the catecholamine synthesis inhibitor α-methyl-p-tyrosine significantly reduced the level of amoebic encystment compared to controls. In conclusion, Acanthamoeba appear to contain a functional adrenergic receptor system of unknown structure which is involved in initiating the encystment process that can be activated and blocked by ß1 agonists and antagonists respectively. Furthermore, the presence of this receptor system in Acanthamoeba indicates that topical ß adrenoceptor blockers may be effective adjunct therapy by reducing the transformation of trophozoites into the highly resistant cyst stage.

Autofluorescence Signatures of Seven Pathogens: Preliminary in Vitro Investigations of a Potential Diagnostic for Acanthamoeba Keratitis.

PURPOSE: Acanthamoeba keratitis can cause devastating damage to the human cornea and is often difficult to diagnose by routine clinical methods. In this preliminary study, we investigated whether Acanthamoeba may be distinguished from other common corneal pathogens through its autofluorescence response. Although only a small number of pathogens were studied, the identification of a unique Acanthamoeba signature would indicate that autofluorescence spectroscopy as a diagnostic method merits further investigation. METHODS: Samples of 7 common pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Elizabethkingia miricola, Achromobacter ruhlandii, Candida albicans, and Acanthamoeba castellanii) in solution were excited with ultraviolet light at a number of successive, narrow wavebands between 260 and 400 nm, and their fluorescence response recorded. Principal Component Analysis was used to allow better visualization of the differences in response to UV light for different species. RESULTS: Acanthamoeba was found to possess a characteristic autofluorescence response and was easily distinguished from E. coli, S. aureus, P. aeruginosa, E. miricola, A. ruhlandii, and C. albicans over a wide range of excitation wavelengths. We also found a clear discrimination between E. coli, C. albicans, and P. aeruginosa at an excitation wavelength of 274 nm, whereas E. miricola, S. aureus, and A. ruhlandii could be separated using an excitation wavelength of 308 nm. CONCLUSIONS: Our results, although preliminary, indicate that autofluorescence spectroscopy shows promise as a diagnostic technique for keratitis. We intend to expand the set of pathogens studied before assessing the feasibility of the technique in vivo by introducing cultures onto pig corneas.

The in vitro efficacy of antimicrobial agents against the pathogenic free-living amoeba Balamuthia mandrillaris.

The free-living amoeba Balamuthia mandrillaris causes usually fatal encephalitis in humans and animals. Only limited studies have investigated the efficacy of antimicrobial agents against the organism. Assay methods were developed to assess antimicrobial efficacy against both the trophozoite and cyst stage of B. mandrillaris (ATCC 50209). Amphotericin B, ciclopirox olamine, miltefosine, natamycin, paromomycin, pentamidine isethionate, protriptyline, spiramycin, sulconazole and telithromycin had limited activity with amoebacidal levels of > 135-500 µM. However, diminazene aceturate (Berenil(®) ) was amoebacidal at 7.8 µM and 31.3-61.5 µM for trophozoites and cysts, respectively. Assays for antimicrobial testing may improve the prognosis for infection and aid in the development of primary selective culture isolation media.

Development of standardized methods for assessing biocidal efficacy of contact lens care solutions against Acanthamoeba trophozoites and cysts.

PURPOSE: To investigate experimental variables in the development of standardized methods to assess the efficacy of contact lens disinfection systems against the trophozoite and cysts of Acanthamoeba spp. METHODS: A. castellanii (ATCC 50370), A. polyphaga (ATCC 30461), and A. hatchetti (CDC: V573) were adapted to axenic culture and used to produce cysts either with Neff's encystment medium (NEM) or starvation on nonnutrient agar (NNA). Challenge test assays and a most probable number approach were used to compare the trophozoite and cysticidal efficacy of four multipurpose disinfectant solutions (MPDSs) and a one-step hydrogen peroxide system (with and without the neutralizing step). RESULTS: With trophozoites, four of four MPDSs and the one-step peroxide system gave ≥3 log10 kill for all strains 6 hours, regardless of culture medium used. Greater resistance was found against cysts, with results for MPDSs varying by species and method of cyst production. Here, 1-3 log10 kill was found with NEM cysts for three of four MPDSs compared with one of four for the NNA cysts at 6 hours (A. castellanii and A. polyphaga, only). The one-step peroxide system gave 1-1.9 log10 kill with NEM cysts and 0.8-1.1 for NNA cysts. Only 3% hydrogen peroxide gave total kill (>3 log10) of NNA cysts at 6 hours. CONCLUSIONS: A reproducible method for determining the susceptibility of Acanthamoeba trophozoites and cysts to contact lens care systems has been developed. This will facilitate assay standardization for assessing the efficacy of such products against the organism and aid development of improved disinfectant and therapeutic agents.

Resistance and growth of Fusarium species in contact lens disinfectant solutions.

PURPOSE: To compare the biocidal efficacy of contact lens care solutions against clinical isolates and the American Type Culture Collection (ATCC) 36031 reference strain of Fusarium species and to determine the ability of the organism to survive and grow in such systems. METHODS: The ISO 14729 reference method to assess biocidal efficacy was applied to the purified conidial form of 10 strains of Fusarium species. Six multipurpose disinfectant solutions (MPDSs), a one-step hydrogen peroxide (Per-1) system, a hydrogen peroxide-sodium chlorite (Per-2) system, and a one-step povidone iodine (PI) system were compared. Challenge organism viability was determined at various intervals, including the manufacturers' recommended disinfection time for the products (4 to 6 hours) and thereafter to 21 days. RESULTS: All MPDSs achieved a 3- to 4-log10 reduction in viability of ATCC 36031 within the recommended disinfection time of 6 hours. For the clinical strains, MPDS-1 (PQ-1 + alexidine) and MPDS-2 (PQ-1 + PHMB) produced 3 to 5 log10 kill after 6 hours. Multipurpose disinfectant solution 3 (PQ-1 + Aldox 0.0006%), MPDS-4 (PQ-1 + Aldox 0.0005% + C-9 ED3A), and MPDS-5 (PQ-1 + Aldox 0.0005%) showed reduced efficacy for the same two strains, with 0.6 to 1.7 log10 kill. Multipurpose disinfectant solution 6 (PHMB) gave 1.6 log10 for one strain and 3 to 4 log10 for the remainder. Growth in all the MPDS was not detected up to 21 days incubation. Per-1 showed less than 1 log10 kill at 6 hours for six of 10 strains, including ATCC 36031, and growth (1.2 to 2.7 log10) occurred with three of 10 strains by 7 days. Per-2 gave less than or equal to 0.5 log10 kill after 6- or 24-hour exposure without growth. The PI system showed 4 to 5 log10 kill for all strains tested by the first time point of 4 hours. However, with the exception of ATCC 36031, growth (1.7 to 4.0 log10) occurred with all strains by 7 days in PI. CONCLUSIONS: All MPDSs were effective against the ATCC 36031 reference strain of Fusarium solani. However, reduced efficacy was found for some MPDSs against the clinical isolates. Unlike MPDSs, peroxide- and povidone iodine-based systems have no continued antimicrobial presence once neutralized, and this can allow growth of surviving Fusarium in the solution. Accordingly, lenses should be subject to fresh disinfection if stored in such solutions for extended periods.

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 MPDS's 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 manufacturer's 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 coxsackievirus, poliovirus and hepatitis A virus.

The antimicrobial activity of simulated solar disinfection (SODIS) against enteric waterborne viruses including coxsackievirus-B5, poliovirus-2 and hepatitis A virus was investigated in this study. Assays were conducted in transparent 12-well polystyrene microtitre plates containing the appropriate viral test suspension. 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 for 6 hours. Aliquots of the viral test suspensions were taken at set time points and the level of inactivation of the viruses was determined by either culture on a HeLa cell monolayer for coxsackievirus-B5 and poliovirus-2 or by utilising a chromogenic antibody-based approach for hepatitis A virus. With coxsackievirus-B5, poliovirus-2 and hepatitis A virus, exposure to SODIS at an optical irradiance of 550 Wm(-2) for 1-2 hours resulted in complete inactivation of each virus. The findings from this study suggest that under appropriate conditions SODIS may be an effective technique for the inactivation of enteric viruses in drinking water. However, further verification studies need to be performed using natural sunlight in the region where the SODIS technology is to be employed to validate our results.

Development of a rapid DNA extraction method and one-step nested PCR for the detection of Naegleria fowleri from the environment.

Naegleria fowleri is a small free-living amoebo-flagellate found in natural and manmade thermal aquatic habitats worldwide. The organism is pathogenic to man causing fatal primary amoebic meningoencephalitis (PAM). Infection typically results from bathing in contaminated water and is usually fatal. It is, therefore, important to identify sites containing N. fowleri in the interests of preventive public health microbiology. Culture of environmental material is the conventional method for the isolation of N. fowleri but requires several days incubation and subsequent biochemical or molecular tests to confirm identification. Here, a nested one-step PCR test, in conjunction with a direct DNA extraction from water or sediment material, was developed for the rapid and reliable detection of N. fowleri from the environment. Here, the assay detected N, fowleri in 18/109 river water samples associated with a nuclear power plant in South West France and 0/10 from a similar site in the UK. Although culture of samples yielded numerous thermophilic free-living amoebae, none were N. fowleri or other thermophilic Naegleria spp. The availability of a rapid, reliable and sensitive one-step nested PCR method for the direct detection of N. fowleri from the environment may aid ecological studies and enable intervention to prevent PAM cases.

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.

Antimicrobial efficacy of multi-purpose contact lens disinfectant solutions following evaporation.

PURPOSE: Non-compliance is a significant factor in contact lens related microbial keratitis and includes solution reuse and failure to recap the lens storage case resulting in evaporation effects. To address this, impact of partial evaporation on the antimicrobial efficacy of multipurpose contact lens care solutions was investigated. METHODS: Solutions were evaporated under a stream of air to 2× and 4× concentration and challenged with Fusarium solani (ATCC 36031), Candida albicans (ATCC 10231) and Acanthamoeba castellanii (ATCC 50370). The level of organism kill at 6h was compared to the non-evaporated product. RESULTS: ReNu with MoistureLoc(®) (RML) lost 90-100% of biocidal activity against C. albicans on evaporation, 75-99% for F. solani and 29-33% with A. castellanii at 2× or 4× concentration, respectively. OPTI-FREE(®) RepleniSH(®) lost 72-90% efficacy against C. albicans and F. solani, and 61% at 2× and 10% at 4× concentration with A. castellanii. ReNu(®) MultiPlus, AQuify(®) Multi-Purpose and Biotrue™ showed only loss in efficacy with C. albicans at 4× concentration giving 79%, 34.5% and 48% reduction, respectively. No loss in biocidal activity on evaporation was obtained with Complete(®) Revitalens for all organisms. CONCLUSION: Partial evaporation can affect biocidal efficacy of multi-purpose solutions and may have been a significant factor in an outbreak of Fusarium keratitis cases associated with RML. Evaporation results in increased binding of cationic disinfectants to counter-ions in the formulation, reducing ability to attach and rupture anionic microbial cell walls. Interaction may also occur between the biocidal ingredient and other components, such as surfactants, resulting in sequestration of activity through micelle formation.

Development of a nested PCR for environmental detection of the pathogenic free-living amoeba Balamuthia mandrillaris.

A DNA extraction and nested PCR method for detecting the pathogenic amoeba Balamuthia mandrillaris from the environment was developed. Sixteen of 17 Californian soil samples were positive compared with 0/44 from the United Kingdom. This approach will enable a greater understanding of B. mandrillaris ecology, geographic distribution, and public health risk.

Antimicrobial activity of simulated solar disinfection against bacterial, fungal, and protozoan pathogens and its enhancement by riboflavin.

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m(-2)) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log(10) after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log(10) after 6 h) was obtained only in the presence of riboflavin and 250 W m(-2) irradiance.

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=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.

The disinfection efficacy of MeniCare soft multipurpose solution against Acanthamoeba and viruses using stand-alone biocidal and regimen testing.

PURPOSE: To assess the disinfection efficacy of MeniCare Soft contact lens multipurpose solution against Acanthamoeba and viruses in suspension and when inoculated on to contact lenses and subjected to rub-and-rinse or no-rub-and-rinse care regimes. METHODS: MeniCare Soft was challenged with Acanthamoeba spp trophozoites or cysts, herpes simplex virus (type 1), adenovirus (type 8), and poliovirus (type 2) and the log reduction in Acanthamoeba viability and viral infectivity determined over time. In addition, contact lenses were incubated with Acanthamoeba and viruses and the number of viable organisms determined after the lenses were processed using rub-and-rinse or no-rub-and-rinse care regimes followed by a 4 hr soaking time. RESULTS: MeniCare Soft showed >3 log reduction against Acanthamoeba spp trophozoites and cysts after 6 hr exposure. Approximately 1 log reduction was found against the 3 viruses after 4 hr exposure. No surviving Acanthamoeba trophozoites or cysts were recovered from any of the contact lens tested when MeniCare Soft was used in a rub-and-rinse or no-rub-and-rinse care regimes (>5.0 log reduction). Rub-and-rinse regimen resulted in a 4.5 to 5.0 log reduction in viruses compared with 3.7 to 5.2 log when no-rub-and-rinse was used. CONCLUSIONS: MeniCare Soft showed effective disinfection efficacy against Acanthamoeba trophozoites and cysts using solution and regimen assays. The viruses were more resistant to disinfection in solution but were removed effectively from contact lenses using a rub-and-rinse or no-rub-and-rinse care regimen.

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.