Determination of the anti-protozoal activity of medicinal agents using the phenomenon of plaque formation by Acanthamoeba spp.

Representatives of the genus Acanthamoeba are among the most widespread protists in the environment. They have a ubiquitous distribution and can sometimes cause quite serious pathologies in humans. The treatment ofp rotozoal infections caused by free-living amoebae is currently limited and often unsuccessful. In the presented investigation, amebicidal activity was determined against both the trophozoites and cysts of Acanthamoeba spp., which were isolated during the microbiological examination of environmental objects. The inhibitory activity of drugs in vitro was determined using the authors' proposed method, which is based on the plaque formation phenomenon: this is initiated by free-living amoebae when cultured in agar containing the bacteria Cellulosimicrobium sp. strain bent-1. Based on a series of experimental studies, the paper proposes a reliable and inexpensive method for determining the anti-protozoal activity of medicinal agents, which will significantly complement the current screening method system when studying existing drugs, or new drugs during their development stage.

Novel anti-Acanthamoeba effects elicited by a repurposed poly (ADP-ribose) polymerase inhibitor AZ9482.

Introduction: Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood. Methods: In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses. Results: Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba trophozoites following AZ9482 treatment. Discussion: Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research.

Amoebicidal effect of chlorine dioxide gas against pathogenic Naegleria fowleri and Acanthamoeba polyphaga.

The pathogenic free-living amoebae, Naegleria fowleri and Acanthamoeba polyphaga, are found in freshwater, soil, and unchlorinated or minimally chlorinated swimming pools. N. fowleri and A. polyphaga are becoming problematic as water leisure activities and drinking water are sources of infection. Chlorine dioxide (ClO2) gas is a potent disinfectant that is relatively harmless to humans at the concentration used for disinfection. In this study, we examined the amoebicidal effects of ClO2 gas on N. fowleri and A. polyphaga. These amoebae were exposed to ClO2 gas from a ready-to-use product (0.36 ppmv/h) for 12, 24, 36, and 48 h. Microscopic examination showed that the viability of N. fowleri and A. polyphaga was effectively inhibited by treatment with ClO2 gas in a time-dependent manner. The growth of N. fowleri and A. polyphaga exposed to ClO2 gas for 36 h was completely inhibited. In both cases, the mRNA levels of their respective actin genes were significantly reduced following treatment with ClO2 gas. ClO2 gas has an amoebicidal effect on N. fowleri and A. polyphaga. Therefore, ClO2 gas has been proposed as an effective agent for the prevention and control of pathogenic free-living amoeba contamination.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

Differential Antimicrobial Efficacy of Preservative-Free Contact Lens Disinfection Systems against Common Ocular Pathogens.

Microbial keratitis is a devastating disease that can cause eye damage and blindness and can be the result of infections by several common ocular pathogens. Importantly, some of these pathogens, such as Acanthamoeba, are particularly unsusceptible to biocides in common contact lens care solutions. Therefore, the disinfection efficacy of preservative-free (PF) disinfection systems against bacteria, fungi, and Acanthamoeba trophozoites and cysts should be assessed as products with the most potential to be efficacious against resistant organisms. PF disinfection systems were analyzed for antimicrobial efficacy. These were the one-step (hydrogen peroxide-based) Clear Care and Clear Care Plus systems and the two-step (povidone-iodine-based) Cleadew system. Stand-alone challenges using bacteria, fungi, and Acanthamoeba were prepared according to the International Standards Organization method 14729. These same challenges were also conducted in the presence of the following contact lenses: Boston RGP, Acuvue Oasys, Biofinity, Ultra, and 2-week PremiO. All challenges were performed at the manufacturer's recommended disinfection time. All preservative-free disinfection systems demonstrated similarly high rates of antimicrobial efficacy when challenged with bacteria or fungi, with or without lenses. However, both Clear Care and Clear Care Plus demonstrated significantly greater disinfection efficacy against Acanthamoeba trophozoites and cysts, with and without lenses (P < 0.05). Cleadew efficacy was impacted by the addition of contact lenses, whereas Clear Care/Clear Care Plus maintained similar efficacies in the absence or presence of lenses. While both hydrogen peroxide and povidone-iodine are highly effective against bacteria and fungi, hydrogen peroxide maintains significantly greater disinfection capabilities than povidone-iodine against all forms of Acanthamoeba. IMPORTANCE Understanding the most efficacious products will allow clinicians to best communicate to patients and consumers the safest products on the market to reduce adverse events, including microbial keratitis, during contact lens use.

Antimicrobial effect of auranofin against Acanthamoeba spp.

Acanthamoebae are opportunistic pathogens that cause serious infections, including Acanthamoeba keratitis, a sight-threatening disease affecting mainly contact lens wearers, and granulomatous amoebic encephalitis, an infection of the central nervous system that occurs mostly in immunocompromised individuals. Although these infections are rare, they are a challenge for healthcare providers. In the last decade, the search for and implementation of novel treatment approaches against these parasites and the infections they cause have intensified, but current options are still unsatisfactory. The aim of this study was to investigate the in vitro activity of the gold-based compound auranofin against Acanthamoeba spp. The study showed that auranofin has potent antimicrobial activity against Acanthamoeba spp., with an IC50 ranging from 2.9 to 3.48 µM, and thus may be useful in the prevention and control of Acanthamoeba infections.

In vitro anti-Acanthamoeba activity of flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and Aconitum napellus.

Acanthamoeba spp. are widely distributed in the environment and cause serious infections in humans. Treatment of Acanthamoeba infections is very challenging and not always effective which requires the development of more efficient drugs against Acanthamoeba spp. The purpose of the present study was to test medicinal plants that may be useful in the treatment of Acanthamoeba spp. Here we evaluated the trophozoital and cysticidal activity of 13 flavonoid glycosides isolated from Delphinium gracile, D. staphisagria, Consolida oliveriana and from Aconitum napellus subsp. Lusitanicum against the amoeba Acanthamoeba castellanii. AlamarBlue Assay Reagent® was used to determine the activity against trophozoites of A. castellanii, and cytotoxic using Vero cells. Cysticidal activity was assessed on treated cysts by light microscopy using a Neubauer chamber to quantify cysts and trophozoites. Flavonoids 1, 2, 3 and 4 showed higher trophozoital activity and selectivity indexes than the reference drug chlorhexidine digluconate. In addition, flavonoid 2 showed 100% cysticidal activity at a concentration of 50 µm, lower than those of the reference drug and flavonoid 3 (100 µm). These results suggest that flavonoids 2 and 3 might be used for the development of novel therapeutic approaches against Acanthamoeba infections after satisfactory in vivo evaluations.

In vitro validation of the amoebicidal activity of commercial eye drops as second activity.

The validation of anti-Acanthamoeba activity of commercial eye drops has gained a great interest recently and a growing number of commercials eye drop were evaluated for their aptitude to inhibit Acanthamoeba as a second pharmacological effect. In the present study, three different eye drops, commercializing in Spain, including TobraDex, Cusimolol and Colircusi antiedema have been tested in vitro against trophozoites and cysts stage of the facultative pathogen Acanthamoeba. The alamarBlue™ method was used to evaluate both trophocidal and cysticidal properties. The most active eye drops were tested for their impact on some programmed cell death features. We found out that the cells inhibition was strain and eye drop dependent, and 5% eye drop was able to eliminate both trophozoite and cyst stage of Acanthamoeba spp. A treatment of 24 h with 5% of TobraDex or Cusimolol was able to show DNA condensation, collapse in the mitochondrial membrane potential and reduction of the ATP level production in Acanthamoeba. We could assume that the present eye drops could induce programed cell death like process in Acanthamoeba via mitochondrial pathway.

Antiamoebic effects of sesquiterpene lactones isolated from the zoanthid Palythoa aff. clavata.

Opportunistic parasitic protozoa of genus Acanthamoeba are responsible to cause severe infections in humans such as Acanthamoeba Keratitis or Amoebic Granulomatous Encephalitis. Current treatments are usually toxic and inefficient and there is a need to access new therapeutic agents. The antiamoebic effects of nephthediol (1) and fourteen germacranolide and eudesmanolide sesquiterpene lactones (2-5, 7-12) isolated from the indigenous zoanthid Palythoa aff. clavata collected at the coast of Lanzarote, Canary Islands were studied against Acanthamoeba castellanii Neff, and the clinical strains A. polyphaga and A. griffini. 4-epi-arbusculin A (11) presented the lowest IC50 value (26,47 ± 1,69 µM) against A. castellanii Neff and low cytotoxicity against murine macrophages, followed by isobadgerin (2), which also showed to be active against A. castellanii Neff cysts. The studies on the mode of action of compounds 2 and 11 revealed these sesquiterpene lactones induce mechanisms of PDC on A. castellanii Neff.

Derivatisation of metronidazole enhances cytotoxic effect against Acanthamoeba genotype T4 isolates and leads to cytomorphological changes in trophozoites.

Amoebae of the genus Acanthamoeba are worldwide distributed causative agents of serious human infections such as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). To date, treatment of these infections is non-uniform and frequently unsuccessful. Recently, the phosphonium salts were studied for their high levels of antimicrobial activity. This work was aimed to investigate the cytotoxic effect of metronidazole and two phosphonium salts (PS1, PS2) on two clinical Acanthamoeba isolates. The isolates showed distinctly higher susceptibility to both phosphonium salts than to metronidazole. The highest susceptibility was noted to PS1 after 48 h of incubation. Metronidazole derivate PS2 showed higher susceptibility than metronidazole. The values of EC50 of PS2 were approximately twenty times lower than EC50 of metronidazole for Acanthamoeba lugdunensis strain and sixteen times lower for Acanthamoeba quina strain after 48 h. Although the therapeutic effect of metronidazole in Acanthamoeba infections is usually insufficient, its derivatisation can result in a significantly higher amoebicidal effect. Cytomorphological changes of trophozoites after exposure to tested compounds included rounding up of the cells, damage of membrane integrity, presence of pathological protrusions, elongation of the cells or pseudocyst-like stages. Obtained results indicate possible therapeutic potential of studied phosphonium salts.

Curcuma longa rhizome extract and Curcumin reduce the adhesion of Acanthamoeba triangularis trophozoites and cysts in polystyrene plastic surface and contact lens.

Curcuma longa and Curcumin have been documented to have a wide spectrum of pharmacological effects, including anti-Acanthamoeba activity. Hence, this study sought to explore the anti-adhesion activity of C. longa extract and Curcumin against Acanthamoeba triangularis trophozoites and cysts in plastic and contact lenses. Our results showed that C. longa extract and Curcumin significantly inhibited the adhesion of A. triangularis trophozoites and cysts to the plastic surface, as investigated by the crystal violet assay (P < 0.05). Also, an 80-90% decrease in adhesion of trophozoites and cysts to the plastic surface was detected following the treatment with C. longa extract and Curcumin at 1/2 × MIC, compared to the control. In the contact lens model, approximately 1 log cells/mL of the trophozoites and cysts was reduced when the cells were treated with Curcumin, when compared to the control. Pre-treatment of the plastic surface with Curcumin at 1/2-MIC reduced 60% and 90% of the adhesion of trophozoites and cysts, respectively. The reduction in 1 Log cells/mL of the adhesion of A. triangularis trophozoites was observed when lenses were pre-treated with both the extract and Curcumin. Base on the results obtained from this study, A. triangularis trophozoites treated with C. longa extract and Curcumin have lost strong acanthopodia, thorn-like projection pseudopodia observed by scanning electron microscope. This study also revealed the therapeutic potentials of C. longa extract and Curcumin, as such, have promising anti-adhesive potential that can be used in the management/prevention of A. triangularis adhesion to contact lenses.

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae.

Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. Since repurposing drugs is an ideal strategy for orphan diseases, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC50 <1 µM) against N. fowleri (n = 19), A. castellanii (n = 12), and B. mandrillaris (n = 27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design.

Nanoparticles based therapeutic efficacy against Acanthamoeba: Updates and future prospect.

Acanthamoeba sp. is a free living amoeba that causes severe, painful and fatal infections, viz. Acanthamoeba keratitis and granulomatous amoebic encephalitis among humans. Antimicrobial chemotherapy used against Acanthamoeba is toxic to human cells and show side effects as well. Infections due to Acanthamoeba also pose challenges towards currently used antimicrobial treatment including resistance and transformation of trophozoites to resistant cyst forms that can lead to recurrence of infection. Therapeutic agents targeting central nervous system infections caused by Acanthamoeba should be able to cross blood-brain barrier. Nanoparticles based drug delivery put forth an effective therapeutic method to overcome the limitations of currently used antimicrobial chemotherapy. In recent years, various researchers investigated the effectiveness of nanoparticles conjugated drug and/or naturally occurring plant compounds against both trophozoites and cyst form of Acanthamoeba. In the current review, a reasonable effort has been made to provide a comprehensive overview of various nanoparticles tested for their efficacy against Acanthamoeba. This review summarizes the noteworthy details of research performed to elucidate the effect of nanoparticles conjugated drugs against Acanthamoeba.

Synthesis, physicochemical properties and biological activities of novel alkylphosphocholines with foscarnet moiety.

A series of alkylphosphocholines with foscarnet moiety was synthesized. The structure of these zwitterionic amphiphiles was modified in both polar and non-polar parts of surfactant molecule. Investigations of physicochemical properties are represented by the determination of critical micelle concentration, the surface tension value at the cmc and the surface area per surfactant head group utilising surface tension measurements. Hydrodynamic diameter of surfactant micelles was determined using the dynamic light scattering technique. Alkylphosphocholines exhibit significant cytotoxic, anticandidal (Candida albicans) and antiamoebal (Acanthamoeba spp. T4 genotype) activity. The relationship between the structure, physicochemical properties and biological activity of the tested compounds revealed that lipophilicity has a significant influence on biological activity of the investigated surfactants. More lipophilic alkylphosphocholines with octadecyl chains show cytotoxic activity against cancer cells which is higher than that of the compounds with shorter alkyl chains. The opposite situation was observed in case of anticandidal and antiamoebal activity of these surfactants. The most active compounds were found to have pentadecyl chains. The foscarnet analogue of miltefosine C15-PFA-C showed the highest anticandidal activity. The minimum value of anticandidal activity of this compound is 1,4 µM thus representing the highest anticandidal activity found within the group of alkylphosphocholines.

Anti-Acanthamoeba disinfection: hands, surfaces and wounds.

Acanthamoebae are facultative parasites causing rare but serious infections such as keratitis and encephalitis and are also known as vectors for several bacterial pathogens, including legionellae and pseudomonads. Acanthamoeba cysts are particularly resilient and enable the amoebae to withstand desiccation and to resist disinfection and therapy. While the search for new therapeutic options has been intensified in the past years, hand and surface disinfectants as well as topical antiseptics for preventing infections have not been studied in detail to date. The aim of this study was to screen well-known and commonly used antimicrobial products in various formulations and different concentrations for their efficacy against Acanthamoeba trophozoites and cysts, including aliphatic alcohols, quaternary ammonium compounds (QACs), peracetic acid (PAA), potassium peroxymonosulfate sulfate (PPMS) and octenidine dihydrochloride (OCT). Of all products tested, OCT and QACs showed the highest efficacy, totally eradicating both trophozoites and cysts within 1 min. The determined 50% effective concentration (EC50) for cysts was 0.196 mg/mL for OCT and 0.119 mg/mL for QACs after 1 min of exposure. PAA and PPMS showed reliable cysticidal efficacies only with prolonged incubation times of 30 min and 60 min, respectively. Aliphatic alcohols generally had limited efficacy, and only against trophozoites. In conclusion, OCT and QACs are potent actives against Acanthamoeba trophozoites and cysts at concentrations used in commercially available products, within contact times suitable for surface and hand disinfection as well as topical antisepsis.

PLGA nanoparticles loaded with Gallic acid- a constituent of Leea indica against Acanthamoeba triangularis.

Acanthamoeba, a genus that contains at least 24 species of free-living protozoa, is ubiquitous in nature. Successful treatment of Acanthamoeba infections is always very difficult and not always effective. More effective drugs must be developed, and medicinal plants may have a pivotal part in the future of drug discovery. Our research focused on investigating the in vitro anti- acanthamoebic potential of Leea indica and its constituent gallic acid in different concentrations. Water and butanol fractions exhibited significant amoebicidal activity against trophozoites and cysts. Gallic acid (100 µg/mL) revealed 83% inhibition of trophozoites and 69% inhibition of cysts. The butanol fraction induced apoptosis in trophozoites, which was observed using tunnel assay. The cytotoxicity of the fractions and gallic acid was investigated against MRC-5 and no adverse effects were observed. Gallic acid was successfully loaded within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with 82.86% encapsulation efficiency, while gallic acid showed 98.24% in vitro release at 48 hours. Moreover, the gallic acid encapsulated in the PLGA nanoparticles exhibited 90% inhibition against trophozoites. In addition, gallic acid encapsulated nanoparticles showed reduced cytotoxicity towards MRC-5 compared to gallic acid, which evidenced that natural product nanoencapsulation in polymeric nanoparticles could play an important role in the delivery of natural products.

Curcuma longa ethanol extract and Curcumin inhibit the growth of Acanthamoeba triangularis trophozoites and cysts isolated from water reservoirs at Walailak University, Thailand.

CURCUMA LONGA: (C. longa) rhizome extract has been traditionally used to treat many infections. Curcumin, a pure compound isolated from the plant, has been documented to possess a wide spectrum of pharmacological effects. The present study aimed to investigate the effects of Thai medicinal plant extracts including C. longa extract and Curcumin on Acanthamoeba triangularis, a causative agent of human Acanthamoeba keratitis. The parasite was isolated from the recreational reservoir at Walailak University, Thailand. The organism was identified as A. triangularis using morphology and 18S rDNA nucleotide sequences. The pathogen was tested for their susceptibility to ethanol extracts of Thai medicinal plants based on eye infection treatment. The ethanol C. longa extract showed the strongest anti-Acanthamoeba activity against both the trophozoites and cysts, followed by Coscinium fenestratum, Coccinia grandis, and Acmella oleracea extracts, respectively. After 24 h, 95% reduction of trophozoite viability was significantly decreased following the treatment with C. longa extract at 125 µg/mL, compared with the control (P < 0.05). The extract at 1,000 µg/mL inhibited 90% viability of Acanthamoeba cyst within 24 h, compared with the control. It was found that the cysts treated with C. longa extract at 500 µg/mL demonstrated abnormal shape after 24 h. The MIC values of C. longa extract and Curcumin against the trophozoites were 125 and 62.5 µg/mL, respectively. While the MICs of the extract and curcumin against the cysts were 500 and 1,000 µg/mL, respectively. The results suggested the potential medicinal benefits of C. longa extract and Curcumin as the alternative treatment of Acanthamoeba infections.

Identification of N-acyl quinolin-2(1H)-ones as new selective agents against clinical isolates of Acanthamoeba keratitis.

A collection of N-substituted quinolin-2(1H)-ones were screened against a panel of clinically relevant protozoa (Leishmania, Trypanosoma and Acanthamoeba). Three quinolin-2(1H)-one compounds were identified as selective anti-Acanthamoeba agents. Further assessment revealed that these compounds were active against both trophozoite and cyst forms of A. castellanii Neff, and caused protozoa death via apoptosis. The data presented herein identify N-acyl quinolin-2(1H)-ones as a promising new class of selective anti-Acanthamoeba agents.

An Original Use of a Bioluminescence Assay to Test the In Vitro Efficacy of Polihexanide in the Eradication of Acanthamoeba Cysts.

PURPOSE: Acanthamoeba keratitis is a rare ocular disease caused by Acanthamoeba spp. The current Acanthamoeba keratitis treatments consist of unlicensed drugs, most commonly polihexanide (PHMB), a biguanide derivative, either as monotherapy or combined with a diamidine. The main purpose of this study was to develop an in vitro bioluminescence assay able to differentiate the antiamoebic activity of PHMB concentrations (0.02%, 0.04%, 0.06%, and 0.08%). Another objective of this study was to evaluate the antiamoebic activity of equal PHMB concentrations at different molecular weights (MWs). METHODS: Adenosine triphosphate (ATP) bioluminescence assay applicability was established by a linear correlation between amoebae number and relative light unit emitted from cysts. The protocol consisted in a series of sequential tests, the first of which compared relative light unit emission and the hemocytometric method after cysts exposure to the different PHMB concentrations. In this assay, dilutions of the initial PHMB concentrations were used as working solutions. Usually, only 5% or less of the instilled dose into the eye is distributed through the cornea; thus, the antiamoebic activity of initial PHMB concentrations 1/10 and 1/100 diluted can reasonably be considered as representative of their undiluted dose administered in vivo. RESULTS: There was no significant difference between the 2 methods. The ATP bioluminescence assay ranked the antiamoebic activity of the working PHMB solutions and showed that the viability of the cysts was equally reduced at fixed PHMB concentration in a wide range of molecular weight. CONCLUSIONS: The ATP bioluminescence assay showed to be a simple and rapid way for characterizing the in vitro antiamoebic activity of working PHMB solutions at different concentrations.

In vitro activity of isavuconazole against three species of Acanthamoeba.

Acanthamoeba keratitis due to a genus of free-living amoebae is a severe corneal infection. Treatment of this disease is based on the combined use of antiseptics and other drugs, including azoles. We tested isavuconazole, the latest marketed azole, in vitro, against A. castellanii, A. lenticulata and A. hatchetti. Our results show that isavuconazole presents slight amoebistatic activity against A. castellanii trophozoites but no cysticidal activity. Isavuconazole could be used only in association for management of AK due to A. castellanii.