Inactivation of Acanthamoeba spp. and Other Ocular Pathogens by Application of Cold Atmospheric Gas Plasma.

Currently there are estimated to be approximately 3.7 million contact lens wearers in the United Kingdom and 39.2 million in North America. Contact lens wear is a major risk factor for developing an infection of the cornea known as keratitis due to poor lens hygiene practices. While there is an international standard for testing disinfection methods against bacteria and fungi (ISO 14729), no such guidelines exist for the protozoan Acanthamoeba, which causes a potentially blinding keratitis most commonly seen in contact lens wearers, and as a result, many commercially available disinfecting solutions show incomplete disinfection after 6 and 24 h of exposure. Challenge test assays based on international standard ISO 14729 were used to determine the antimicrobial activity of cold atmospheric gas plasma (CAP) against Pseudomonas aeruginosa, Candida albicans, and trophozoites and cysts of Acanthamoeba polyphaga and Acanthamoeba castellanii P. aeruginosa and C. albicans were completely inactivated in 0.5 min and 2 min, respectively, and trophozoites of A. polyphaga and A. castellanii were completely inactivated in 1 min and 2 min, respectively. Furthermore, for the highly resistant cyst stage of both species, complete inactivation was achieved after 4 min of exposure to CAP. This study demonstrates that the CAP technology is highly effective against bacterial, fungal, and protozoan pathogens. The further development of this technology has enormous potential, as this approach is able to deliver the complete inactivation of ocular pathogens in minutes, in contrast to commercial multipurpose disinfecting solutions that require a minimum of 6 h.

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.

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.

Correlation of ex vivo and in vivo confocal microscopy imaging of Acanthamoeba.

BACKGROUND/AIMS: The aim of this study was to correlate the various forms of Acanthamoeba on ex vivo confocal microscopy (EVCM) with in vivo confocal microscopy (IVCM) and findings from cultured positive cases of Acanthamoeba keratitis. METHODS: Acanthamoeba live, dead and empty cysts, and live trophozoites were prepared in vitro and inoculated into porcine cornea using a sterile 26-gauge needle and examined ex vivo using the Heidelberg Retina Tomograph II/Rostock Corneal Module. IVCM images from 12 cultured positive Acanthamoeba cases, obtained using the same instrument, were compared with EVCM findings. Phase contrast images were also obtained to compare with both EVCM and IVCM findings. The change in cyst morphology with depth was evaluated by imaging the same cysts over a defined cornea depth measurement. RESULTS: EVCM morphologies for live cysts included four main types-hyper-reflective central dot with hyper-reflective outer ring, hyper-reflective central dot with hyporeflective outer region, stellate shaped hyper-reflective centre with hyporeflective outer region and hyper-reflective round/polygonal shaped cyst; one main type for dead cysts-hyper-reflective central dot with hyporeflective outer region; two main types for empty cysts- hyper-reflective central dot with hyper-reflective outer ring/hyporeflective outer region; and one main type for trophozoites-large coarse speckled area of heterogeneous hyper-reflective material. Matching IVCM images show good correlation with EVCM. Cyst morphology altered when imaged at different depths. CONCLUSION: EVCM demonstrated the various forms of Acanthamoeba cyst and trophozoites can be used as a reference to identify similar structures on IVCM.

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 Investigation of Thymol Formulations Containing Poloxamer 407 and Hydroxypropyl Methylcellulose to Inhibit Candida Biofilm Formation and Demonstrate Improved Bio-Compatibility.

The aim of this study was to investigate the potential of thymol to inhibit Candida biofilm formation and improve thymol biocompatibility in the presence of hydroxypropyl methylcellulose (HPMC) and poloxamer 407 (P407), as possible drug carriers. Thymol with and without polymers were tested for its ability to inhibit biofilm formation, its effect on the viability of biofilm and biocompatibility studies were performed on HEK 293 (human embryonic kidney) cells. Thymol showed a concentration dependent biofilm inhibition; this effect was slightly improved when it was combined with HPMC. The Thymol-P407 combination completely inhibited the formation of biofilm and the antibiofilm effect of thymol decreased as the maturation of Candida biofilms increased. The effect of thymol on HEK 293 cells was a loss of nearly 100% in their viability at a concentration of 250 mg/L. However, in the presence of P407, the viability was 25% and 85% using neutral red uptake and sulforhodamine B assays, respectively. While, HPMC had less effect on thymol activity the thymol-P407 combination showed a superior inhibitory effect on biofilm formation and better biocompatibility with human cell lines. The combination demonstrates a potential medical use for the prevention of Candida biofilm formation.

The Activity of PHMB and Other Guanidino Containing Compounds against Acanthamoeba and Other Ocular Pathogens.

In recent years, a rise in the number of contact lens users in the UK and worldwide coincided with an increased incidence of microbial keratitis. The aim of this study was to investigate the antimicrobial activities of polyhexamethylene guanidine (PHMG), polyaminopropyl biguanide (PAPB), and guazatine in comparison to the common contact lens disinfectant constituent, polyhexamethylene biguanide (PHMB). The study investigated these compounds against a broad range of organisms, including Acanthamoeba castellanii, Acanthamoeba polyphaga, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. This study demonstrated that PHMG, PAPB, and guazatine are equal in activity to PHMB against Acanthamoeba trophozoites and cysts. PHMG and PAPB are also equal in activity to PHMB against S. aureus and P. aeruginosa, whereas PHMG shows significantly better activity than PHMB against C. albicans (p < 0.001). To our knowledge, this is the first study to demonstrate the effectiveness of PHMB, PHMG, PAPB, and guazatine against Acanthamoeba and other ocular pathogens. As alternatives to PHMB, these compounds warrant further investigation for inclusion in contact lens solutions and for the treatment of keratitis.

Thymoquinone: Hydroxypropyl-ß-cyclodextrin Loaded Bacterial Cellulose for the Management of Wounds.

The need for more advantageous and pharmaceutically active wound dressings is a pressing matter in the area of wound management. In this study, we explore the possibility of incorporating thymoquinone within bacterial cellulose, utilising cyclodextrins as a novel method of solubilising hydrophobic compounds. The thymoquinone was not soluble in water, so was incorporated within hydroxypropyl-ß-cyclodextrin before use. Thymoquinone: hydroxypropyl-ß-cyclodextrin inclusion complex produced was found to be soluble in water up to 7% (w/v) and was stable with no crystal formation for at least 7 days with the ability to be loaded within the bacterial cellulose matrix. The inclusion complex was found to be thermally stable up to 280 °C which is far greater than the production temperature of 80 °C and was stable in phosphate-buffered saline and extraction solvents in permeation and dose experiments. The adhesion properties of the Thymoquinone: hydroxypropyl-ß-cyclodextrin loaded bacterial cellulose dressings were tested and found to be 2.09 N. Permeation studies on skin mimicking membrane Strat-M showed a total permeated amount (0-24 h) of 538.8 µg cm-2 and average flux after a 2 h lag of 22.4 µg h-1 cm-2. To the best of our knowledge, the methods outlined in this study are the first instance of loading bacterial cellulose with thymoquinone inclusion complex with the aim of producing a pharmaceutically active wound dressing.

The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a 'redox-switch' in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.

The Mould War: Developing an Armamentarium against Fungal Pathogens Utilising Thymoquinone, Ocimene, and Miramistin within Bacterial Cellulose Matrices.

An increase in antifungal resistance has seen a surge in fungal wound infections in patients who are immunocompromised resulting from chemotherapy, disease, and burns. Human pathogenic fungi are increasingly becoming resistant to a sparse repertoire of existing antifungal drugs, which has given rise to the need to develop novel treatments for potentially lethal infections. Bacterial cellulose (BC) produced by Gluconacetobacter xylinus has been shown to possess many properties that make it innately useful as a next-generation biopolymer to be utilised as a wound dressing. The current study demonstrates the creation of a pharmacologically active wound dressing by loading antifungal agents into a biopolymer hydrogel to produce a novel wound dressing. Amphotericin B is known to be highly hepatotoxic, which reduces its appeal as an antifungal drug, especially in patients who are immunocompromised. This, coupled with an increase in antifungal resistance, has seen a surge in fungal wound infections in patients who are immunodeficient due to chemotherapy, disease, or injury. Antifungal activity was conducted via Clinical & Laboratory Standards Institute (CLSI) M27, M38, M44, and M51 against Candida auris, Candida albicans, Aspergillus fumigatus, and Aspergillus niger. This study showed that thymoquinone has a comparable antifungal activity to amphotericin B with mean zones of inhibition of 21.425 ± 0.925 mm and 22.53 ± 0.969 mm, respectively. However, the mean survival rate of HEp-2 cells when treated with 50 mg/L amphotericin B was 29.25 ± 0.854% compared to 71.25 ± 1.797% when treated with 50 mg/L thymoquinone. Following cytotoxicity assays against HEp-2 cells, thymoquinone showed a 71.25 ± 3.594% cell survival, whereas amphotericin B had a mean cell survival rate of 29.25 ± 1.708%. The purpose of this study was to compare the efficacy of thymoquinone, ocimene, and miramistin against amphotericin B in the application of novel antifungal dressings.

Recent Advances and Applications of Bacterial Cellulose in Biomedicine.

Bacterial cellulose (BC) is an extracellular polymer produced by Komagateibacter xylinus, which has been shown to possess a multitude of properties, which makes it innately useful as a next-generation biopolymer. The structure of BC is comprised of glucose monomer units polymerised by cellulose synthase in ß-1-4 glucan chains which form uniaxially orientated BC fibril bundles which measure 3-8 nm in diameter. BC is chemically identical to vegetal cellulose. However, when BC is compared with other natural or synthetic analogues, it shows a much higher performance in biomedical applications, potable treatment, nano-filters and functional applications. The main reason for this superiority is due to the high level of chemical purity, nano-fibrillar matrix and crystallinity. Upon using BC as a carrier or scaffold with other materials, unique and novel characteristics can be observed, which are all relatable to the features of BC. These properties, which include high tensile strength, high water holding capabilities and microfibrillar matrices, coupled with the overall physicochemical assets of bacterial cellulose makes it an ideal candidate for further scientific research into biopolymer development. This review thoroughly explores several areas in which BC is being investigated, ranging from biomedical applications to electronic applications, with a focus on the use as a next-generation wound dressing. The purpose of this review is to consolidate and discuss the most recent advancements in the applications of bacterial cellulose, primarily in biomedicine, but also in biotechnology.

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.

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.

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.

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.

Mechanical performance of additively manufactured pure silver antibacterial bone scaffolds.

Implant infection is a serious complication resulting in pain, mortality, prolonged recovery, and antimicrobial resistance (AMR). Reducing the risk-of-infection associated with tissue implants require imminent attention, where pure silver (Ag) offers enormous potential. However, the printability, mechanical performance nor microbial resistance of additively manufactured (AM) pure Ag is unavailable in literature. This is critical as Ag is thought to play a vital role in the development of AM patient-specific infection resistant implants in the decade to come. The study therefore additively manufactured 99.9% pure-Ag through selective laser melting (SLM) and systematically investigates its mechanical performance. The validated SLM process parameters were then used to conceive two fully porous bone scaffold each at approximately 68 and 90% (wt.) porosity. While the study brings to attention the potential defects in SLM pure-Ag through X-ray nanotomography (X-ray nCT), the mechanical properties of porous Ag scaffolds were found to be similar to cancellous bone. The study achieved the highest SLM pure-Ag density of 97% with Young's modulus (E), elastic limit (σe), yield strength (σy), ultimate strength (σult) and ultimate strain (εult) in the range of 15.5-17.8 GPa, 50.7-57.7 MPa, 57.6-67.2 MPa, 82.4-95.9 MPa and 0.07-0.10 respectively. The antimicrobial efficacy of printed silver was tested against the common implant infection-causing Staphylococcus aureus and led to 90% and 99.9% kill in 4 and 14 h respectively. The study, therefore, is a first step towards achieving a new generation Ag-based AM infection resistant porous implants.

In Vitro Evaluation of the Inhibitory Effect of Topical Ophthalmic Agents on Acanthamoeba Viability.

PURPOSE: To compare the antimicrobial effect of topical anesthetics, antivirals, antibiotics, and biocides on the viability of Acanthamoeba cysts and trophozoites in vitro. METHODS: Amoebicidal and cysticidal assays were performed against both trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370) and Acanthamoeba polyphaga (ATCC 30461). Test agents included topical ophthalmic preparations of common anesthetics, antivirals, antibiotics, and biocides. Organisms were exposed to serial two-fold dilutions of the test compounds in the wells of a microtiter plate to examine the effect on Acanthamoeba spp. In addition, the toxicity of each of the test compounds was determined against a mammalian cell line. RESULTS: Proxymetacaine, oxybuprocaine, and especially tetracaine were all toxic to the trophozoites and cysts of Acanthamoeba spp., but lidocaine was well tolerated. The presence of the benzalkonium chloride (BAC) preservative in levofloxacin caused a high level of toxicity to trophozoites and cysts. With the diamidines, the presence of BAC in the propamidine drops was responsible for the activity against Acanthamoeba spp. Hexamidine drops without BAC showed good activity against trophozoites, and the biguanides polyhexamethylene biguanide, chlorhexidine, alexidine, and octenidine all showed excellent activity against trophozoites and cysts of both species. CONCLUSIONS: The antiamoebic effects of BAC, povidone iodine, and tetracaine are superior to the current diamidines and slightly inferior to the biguanides used in the treatment for Acanthamoeba keratitis. TRANSLATIONAL RELEVANCE: Ophthalmologists should be aware that certain topical anesthetics and ophthalmic preparations containing BAC prior to specimen sampling may affect the viability of Acanthamoeba spp. in vivo, resulting in false-negative results in diagnostic tests.

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.

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.

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 micro g/ml), chlorhexidine digluconate (30 micro g/ml), myristamidopropyl dimethylamine (20 micro g/ml), H(2)O(2) (3%), and two multipurpose contact lens solutions (MPS-1 and MPS-2, based on 1 micro g 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 MgCl(2) 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 degrees 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.