Wayne Heaselgrave

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.

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.

Disinfection efficacy and encystment rate of soft contact lens multipurpose solutions against Acanthamoeba.

Objectives: To assess the ability of commercial and experimental soft contact lens multipurpose solutions (MPS) to promote Acanthamoeba trophozoite encystment and their biocidal efficacy against Acanthamoeba trophozoites and cysts. The effects on encystment and disinfection efficacy by the incorporation of propylene glycol (PG) in the formulation of MPS were also investigated. Methods: Acanthamoeba trophozoites (Acanthamoeba castellanii ATCC 50730 and Acanthamoeba polyphaga CCAP 1501/3G) were inoculated into MeniCare Soft, MeniCare Soft (−PG), Epica Cold II, OptiFree Plus, and Rohto C cube Softone—Moist MPS, and the percentage of encystment induced by each solution was determined after 24 hr. In addition, Acanthamoeba trophozoites and cysts (A. castellanii ATCC 50730 and A. polyphaga CCAP 1501/3G and Ros) were also inoculated into each of the five MPS, and their log reduction determined after 0, 1, 4, 6, and 24 hr of incubation using stand-alone assays. Results: Significantly higher encystment rates were found with Epica Cold II for A. polyphaga CCAP 1501/3G and Rohto C cube Softone—Moist for A. polyphaga and A. castellanii compared with the other MPS assessed (P<0.05). MeniCare Soft, MeniCare Soft (−PG), and Opti-Free Plus produced little or no encystment, with mean encystment values ranging from 0.0% to 2.0%. A significantly higher disinfection efficacy was found with MeniCare Soft, MeniCare Soft (−PG), and Epica Cold II compared with Opti-Free Plus and Rohto C cube Softone—Moist (P<0.05). Conclusions: Significant differences in encystment rate and disinfection efficacy between MPS were found. The presence of PG in the formulation of MeniCare Soft did not affect the disinfection efficacy or the encystment rate. The latter indicates that other factors play a role in the induction of Acanthamoeba encystment after inoculation into MPS.

Antimicrobial Activity of Simulated Solar Disinfection against Bacterial, Fungal, and Protozoan Pathogens and Its Enhancement by Riboflavin

ABSTRACT 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 log10 after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log10 after 6 h) was obtained only in the presence of riboflavin and 250 W m−2 irradiance.

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.

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.

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.