Acanthamoeba spp.: Efficacy of Bioclen FR One Step®, a povidone-iodine based system for the disinfection of contact lenses

Pathogenic strains of Acanthamoeba are causative agents of a serious sight-threatening infection of the eye known as Acanthamoeba keratitis. The prevalence of this infection has risen in the past 20 years, mainly due to the increase in number of contact lens wearers. Bioclen FR One Step® (Ophtecs Corporation) is the only available povidone-iodine based system for the disinfection of silicone hydrogel lenses and soft contact lenses on the market. Bioclen FR has been proven to be highly effective against bacteria and fungi that can cause problems for contact lens users. In this study, Bioclen FR One Step® was tested against three clinical Acanthamoeba isolates from contact lens cases. The results demonstrated that the tested Acanthamoeba clinical strains were sensitive to Bioclen FR One Step®.     

First report of a mixed infection due to Acanthamoeba genotype T3 and Vahlkampfia in a cosmetic soft contact lens wearer in Iran

Acanthamoeba keratitis cases have emerged in the recent years in Iran. In this case, an amoebic keratitis due to a mixed infection with Acanthamoeba and Vahlkampfia species is reported. Corneal scrapes, contact lenses and contact lens cases obtained from the patient were analysed and were positive for cysts of Acanthamoeba and Vahlkampfia genera. Genus-specific PCR was carried out for both genera, confirming the microscopic observations. To our knowledge, this is the first reported case of a possible mixed amoebic infection due to Acanthamoeba and Vahlkampfia and raises awareness within contact lens wearers in Iran.     

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.     

Flow Cytometry for Determination of the Efficacy of Contact Lens Disinfecting Solutions against Acanthamoeba spp.

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10⁵ to 10⁶/ml) at 22°C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.     

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.     

Acanthamoeba castellanii: In vitro effects of selected biological, physical and chemical factors

Trophozoites and cysts of free-living Acanthamoeba castellanii present a serious risk to human health as causative agents of human diseases such as fatal granulomatous amoebic encephalitis and Acanthamoeba keratitis that is reported from various part of the world, also in Poland, with increasing frequency, particularly in the contact lens wearers. The amphizoic amoebae are generally extremely resistant to different chemical agents, however, several strains/isolates within A. castellanii may differ in virulence. Among the features considered as associated with the amoeba pathogenicity, temperature tolerance and resistance to different environmental conditions are reported. In the present study, A. castellanii strain cultured in 26 °C after several year passages were tested for sensibility/tolerance to instant temperature changes as well as exposition to deuterium oxide, D₂O. Significant decrease of number of viable amoebae during in vitro exposition to D₂O occurred, but no changes in trophozoites/cysts ratio. The ability of the strain examined to develop in higher temperature may indicate a wide adaptation reserve and its pathogenic potential.     

Antineoplastic kinase inhibitors: A new class of potent anti-amoebic compounds

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300–400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC₅₀ values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease.     

In vitro amoebicidal activity of Origanum syriacum and Origanum laevigatum on Acanthamoeba castellanii cysts and trophozoites

In some patients, complete treatment of amoebic keratitis is difficult because of the resistance of cysts to therapeutic agents. The aim of this study was to evaluate the in vitro amoebicidal activity of methanolic extracts of Origanum syriacum and Origanum laevigatum. In the presence of methanolic extracts (ranging from 1.0 to 32.0mg/ml), numbers of the viable Acanthamoeba castellanii trophozoites and cysts were decreased. Both extracts showed a time and dose dependent amoebicidal action on the trophozoites and cysts. Of the extracts tested, O. syriacum showed the stronger amoebicidal effect on the trophozoites and cysts. In the presence of 32 mg/ml extract, no viable trophozoites were observed within third hour. The extract was also found effective against the cysts within 24th hour. In the case of O. laevigatum, no viable trophozoites were observed within 72nd hour at the concentrations of 16 and 32 mg/ml. As expected, cysts were found more resistant to the extracts than the trophozoites.     

Controlled Release Matrix Tablets of Olanzapine: Influence of Polymers on the <Emphasis Type="Italic">In Vitro</Emphasis> Release and Bioavailability

Controlled-release (CR) tablet formulation of olanzapine was developed using a binary mixture of Methocel® K100 LV-CR and Ethocel® standard 7FP premium by the dry granulation slugging method. Drug release kinetics of CR tablet formulations F1, F2, and F3, each one suitably compressed for 9-, 12-, and 15-kg hardness, were determined in a dissolution media of 0.1 N HCl (pH 1.5) and phosphate buffer (pH 6.8) using type II dissolution apparatus with paddles run at 50 rpm. Ethocel® was found to be distinctly controlling drug release, whereas the hardness of tablets and pH of the dissolution media did not significantly affect release kinetics. The CR test tablets containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness exhibited pH-independent zero-order release kinetics for 24 h. In vivo performance of the CR test tablet and conventional reference tablet were determined in rabbit serum using high-performance liquid chromatography coupled with electrochemical detector. Bioavailability parameters including C_max, T_max, and AUC_0–48 h of both tablets were compared. The CR test tablets produced optimized C_max and extended T_max (P < 0.05). A good correlation of drug absorption in vivo and drug release in vitro (R² = 0.9082) was observed. Relative bioavailability of the test tablet was calculated as 94%. The manufacturing process employed was reproducible and the CR test tablets were stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. It was concluded that the CR test tablet formulation successfully developed may improve tolerability and patient adherence by reducing adverse effects.Key words: bioavailability, controlled release, Ethocel®, olanzapine     

Formulation of Microemulsion Systems for Dermal Delivery of Silymarin

Silymarin is a standardized extract from Silybum marianum seeds, known for its many skin benefits such as antioxidant, anti-inflammatory, and immunomodulatory properties. In this study, the potential of several microemulsion formulations for dermal delivery of silymarin was evaluated. The pseudo-ternary phase diagrams were constructed for the various microemulsion formulations which were prepared using glyceryl monooleate, oleic acid, ethyl oleate, or isopropyl myristate as the oily phase; a mixture of Tween 20®, Labrasol®, or Span 20® with HCO-40® (1:1 ratio) as surfactants; and Transcutol® as a cosurfactant. Oil-in-water microemulsions were selected to incorporate 2% w/w silymarin. After six heating–cooling cycles, physical appearances of all microemulsions were unchanged and no drug precipitation occurred. Chemical stability studies showed that microemulsion containing Labrasol® and isopropyl myristate stored at 40°C for 6 months showed the highest silybin remaining among others. The silybin remainings depended on the type of surfactant and were sequenced in the order of: Labrasol® > Tween 20® > Span 20®. In vitro release studies showed prolonged release for microemulsions when compared to silymarin solution. All release profiles showed the best fits with Higuchi kinetics. Non-occlusive in vitro skin permeation studies showed absence of transdermal delivery of silybin. The percentages of silybin in skin extracts were not significantly different among the different formulations (p > 0.05). Nevertheless, some silybin was detected in the receiver fluid when performing occlusive experiments. Microemulsions containing Labrasol® also were found to enhance silymarin solubility. Other drug delivery systems with occlusive effect could be further developed for dermal delivery of silymarin.Key words: dermal delivery, microemulsion, silybin, silymarin     

Phosphonate fertilizers suppressed root knot nematodes Meloidogyne javanica and M. incognita

The efficacy of the phosphonate fertilizers, Calphos^® (a.i. calcium phosphonate), Magphos^® (a.i. magnesium phosphonate and potassium phosphonate) and Phosphoros^® (a.i. potassium phosphonate) against two species of root knot nematodes (RKN), Meloidogyne javanica and M. incognita is evaluated. Laboratory experiments showed that Calphos^®, Magphos^® and their main components inhibited egg hatching and caused 100% mortality of the second stage juveniles (J2s) of the two RKN species; the hatching inhibition effects persisted after transferring the egg masses of both species to water. However, Phosphoros^® (0.5%) did not suppress egg hatching or the survival of J2s of both RKN species. No hatching occurred when egg masses were treated for one week with the nematicide Vydate L^® (2 ml/l), however, J2s hatched when the Vydate L^® treated egg masses were moved to water. The glasshouse study indicated that Magphos^®, Calphos^® and Phosphoros^® reduced root galling caused by M. javanica by 98, 66 and 47%, respectively, in comparison to the untreated controls. Magphos^® resulted in the lowest number of root galls formed by M. incognita, the reduction was 84%. In contrast, Calphos^® and Phosphoros^® reduced galling by 47 and 39%, respectively. The Magphos^® treatment resulted in the lowest numbers of egg masses and the lowest reproductive factor (RF) of both nematode species. However, plants treated with Phosphoros^® resulted in higher foliage weights compared with the application of the other two fertilizers and the untreated plants.     

Nanoemulsions Containing a Coumarin-Rich Extract from <Emphasis Type="Italic">Pterocaulon balansae</Emphasis> (Asteraceae) for the Treatment of Ocular <Emphasis Type="Italic">Acanthamoeba</Emphasis> Keratitis

This study describes the incorporation of a coumarin-rich extract from Pterocaulon balansae into nanoemulsions intended for the local treatment of ocular keratitis caused by Acanthamoeba. The n-hexane dewaxed extract of P. balansae was characterized by HPLC/PDA and UPLC/MS. The presence of four major coumarins was detected, where 5-methoxy-6,7-methylenedioxycoumarin was selected as a chemical marker. This extract was then incorporated into nanoemulsions composed of medium chain triglycerides and egg-lecithin, through spontaneous emulsification. Such a procedure yielded the formation of monodisperse nanoemulsions in a sub-300-nm range, regardless of the amount of extract incorporated (1.0–5.0 mg/mL). The amoebicidal activity against Acanthamoeba castellanii was both dose-dependent and incubation time-dependent. A reduction of 95% of trophozoite viability was detected after 24 h of incubation with a nanoemulsion at 1.25 mg/mL of coumarins, being a similar effect detected for chlorhexidine. These results suggest a potential of the formulations developed in this study as a new strategy for the treatment of ocular keratitis caused by Acanthamoeba.     

Development of Controlled-Release Matrix Tablet of Risperidone: Influence of Methocel®- and Ethocel®-Based Novel Polymeric Blend on <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Bioavailability

Controlled-release (CR) matrix tablet of 4 mg risperidone was developed using flow bound dry granulation–slugging method to improve its safety profile and compliance. Model formulations F1, F2, and F3, consisting of distinct blends of Methocel® K100 LV-CR and Ethocel® standard 7FP premium, were slugged. Each batch of granules (250–1,000 μm), obtained by crushing the slugs, was divided into three portions after lubrication and then compressed to 9-, 12-, and 15-kg hard tablets. In vitro drug release studies were carried out in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8) using a paddle dissolution apparatus run at 50 rpm. The CR test tablet, containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness, exhibited pH-independent zero-order release kinetics for 24 h. The drug release rate was inversely proportional to the content of Ethocel®, while the gel layer formed of Methocel® helped in maintaining the integrity of the matrix. Changes in the hardness of tablet did not affect the release kinetics. The tablets were reproducible and stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. Risperidone and its active metabolite, 9-hydroxyrisperidone, present in the pooled rabbit’s serum, were analyzed with HPLC-UV at λ_max 280 nm. The CR test tablet exhibited bioequivalence to reference conventional tablet in addition to the significantly (p < 0.05) optimized peak concentration, C_max, and extended peak time, T_max, of the active moiety. There was a good association between drug absorption in vivo and drug release in vitro (R² = 0.7293). The successfully developed CR test tablet may be used for better therapeutic outcomes of risperidone.KEY WORDS: controlled release, dry granulation slugging method, risperidone     

Characterization of growth and Oryctes rhinoceros nudivirus production in attached cultures of the DSIR-HA-1179 coleopteran insect cell line

The DSIR-HA-1179 coleopteran cell line is a susceptible and permissive host to the Oryctesrhinoceros nudivirus (OrNV), which has been used as a biocontrol agent against the coconut rhinoceros beetle (Oryctes rhinoceros); a pest of palms in the Asia-Pacific region. However, little is known about growth and metabolism of this cell line, knowledge of which is necessary to develop an in vitro large-scale OrNV production process. The strong anchorage-dependent characteristics of the cell line, its particular fragility and its tendency to form dense clumps when manipulated, are the most likely reasons that have precluded further development of the cell line. In order to characterize DSIR-HA-1179 cells, there was first a need for a reliable technique to count the cells. A homogenous cell suspension suitable for enumeration could be produced by treatment with TrypLE Express™ with optimum mean time for cell release calculated as 30 min. The cell line was adapted to grow in four serum-supplemented culture media namely TC-100, IPL-41, Sf-900 II and Sf-900 III and cell growth, glucose consumption, lactate and ammonia production were assessed from static-batch cultures. The maximum viable cell density was reached in Sf-900 II (17.9 × 10⁵ cells/ml), with the maximum specific growth rate observed in this culture medium as well (0.0074 h⁻¹). Higher production of OrNV was observed in IPL-41 and TC-100 (4.1 × 10⁷ TCID₅₀/ml) than in cultures infected in Sf-900 III (2.0 × 10⁷ TCID₅₀/ml) and Sf-900 II (1.4 × 10⁷ TCID₅₀/ml). At the end of the growth period, glucose was completely consumed in cultures grown in TC-100, while remained in excess in the other three culture media. The cell line produced lactate and ammonia to very low levels in the TC-100 culture medium which is a promising aspect for its cultivation at large-scale.     

Positively Charged Polymeric Nanoparticle Reservoirs of Terbinafine Hydrochloride: Preclinical Implications for Controlled Drug Delivery in the Aqueous Humor of Rabbits

Frequent instillation of terbinafine hydrochloride (T HCl) eye drops (0.25%, w/v) is necessary to maintain effective aqueous humor concentrations for treatment of fungal keratitis. The current approach aimed at developing potential positively charged controlled-release polymeric nanoparticles (NPs) of T HCl. The estimation of the drug pharmacokinetics in the aqueous humor following ocular instillation of the best-achieved NPs in rabbits was another goal. Eighteen drug-loaded (0.50%, w/v) formulae were fabricated by the nanopreciptation method using Eudragit® RS100 and chitosan (0.25%, 0.5%, and 1%, w/v). Soybean lecithin (1%, w/v) and Pluronic® F68 (0.5%, 1%, and 1.5%, w/v) were incorporated in the alcoholic and aqueous phases, respectively. The NPs were evaluated for particle size, zeta potential, entrapment efficiency percentage (EE%), morphological examination, drug release in simulated tear fluid (pH 7.4), Fourier-transform IR (FT-IR), X-ray diffraction (XRD), physical stability (2 months, 4°C and 25°C), and drug pharmacokinetics in the rabbit aqueous humor relative to an oily drug solution. Spherical, discrete NPs were successfully developed with mean particle size and zeta potential ranging from 73.29 to 320.15 nm and +20.51 to +40.32 mV, respectively. Higher EE% were achieved with Eudragit® RS100-based NPs. The duration of drug release was extended to more than 8 h. FT-IR and XRD revealed compatibility between inactive formulation ingredients and T HCl and permanence of the latter’s crystallinity, respectively. The NPs were physically stable, for at least 2 months, when refrigerated. F5-NP suspension significantly (P < 0.05) increased drug mean residence time and improved its ocular bioavailability; 1.657-fold.Key words: aqueous humor, chitosan, Eudragit® RS100, nanoparticles, terbinafine hydrochloride     

Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City

A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis.     

Rapid identification of Acanthamoeba from contact lens case using loop-mediated isothermal amplification method

A method employing loop-mediated isothermal amplification (LAMP) of 18S ribosomal RNA gene was developed to detect Acanthamoeba in contact lens cases. A prevalence of 7% (10/150) was detected, with 100% sensitivity and 100% specificity when compared with the standard culture technique. Using visual inspection of turbidity a minimum of 10 pg of Acanthamoeba DNA could be detected, 10 times more sensitive than quantitative PCR employing two of the LAMP primers. The production of LAMP amplicons was confirmed by gel-electrophoresis and ethidium bromide staining. The LAMP procedure takes less than 2 h to perform and will be useful for incorporation into a point-of-care screening of suspected Acanthamoeba infection.     

Acanthamoeba adherence to contact lenses, removal by rinsing procedures, and survival to some ophthalmic products.

Unworn soft and rigid gas-permeable contact lenses were inoculated with an Acanthamoeba keratitis strain to study the protozoon's ability to adhere. Furthermore, the efficacy of the rinsing in saline on acanthamoeba removal was evaluated, as well as the amebicidal activity of five commercial cleaning/disinfecting products: hydrogen peroxide, chlorhexidine, polyaminopropyl biguanide-poloxamine, thimerosal-polyquaternium, and thimerosal-chlorhexidine. Microscopic count of cells showed that Acanthamoeba trophozoites and cysts adhered to all types of contact lenses. A significantly greater adherence of trophozoites than cysts was recorded. The rinsing in saline using a flow-method was significantly more effective than the immersion-method, particularly in removing trophozoites from rigid gas-permeable lenses. The cleaning/disinfecting solutions tested were ineffective in removing or in affecting the viability of all Acanthamoeba trophozoites or cysts in the 17 hours allotted for the experiment. The need for a better care in mechanical and physical hygiene procedures is stressed.     

Apoptosis of Acanthamoeba castellanii Trophozoites Induced by Oleic Acid

Acanthamoeba spp. can be parasitic in certain situations and are responsible for serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis, and cutaneous acanthamoebiasis. We analyzed the fatty acid composition of Acanthamoeba castellanii trophozoites and tested the inhibitory activity of the main fatty acids, oleic acid and arachidonic acid, in vitro. Oleic acid markedly inhibited the growth of A. castellanii, with trophozoite viability of 57.4% at a concentration of 200 μM. Caspase‐3 staining and annexin V assays showed that apoptotic death occurred in A. castellanii trophozoites. Quantitative PCR and dot blot analysis showed increased levels of metacaspase and interleukin‐1β converting enzyme, which is also an indication of apoptosis. In contrast, arachidonic acid showed negligible inhibition of growth of A. castellanii trophozoites. Stimulated expression of Atg3, Atg8 and LC3A/B genes and monodansylcadaverine labeling suggested that oleic acid induces apoptosis by triggering autophagy of trophozoites.