Acanthamoeba Hatchetti Research Articles

Efficacy of Off-Label Anti-Amoebic Agents to Suppress Trophozoite Formation of Acanthamoeba spp. on Non-Nutrient Agar Escherichia Coli Plates.

Acanthamoeba keratitis (AK) is a dangerous infectious disease, which is associated with a high risk of blindness for the infected patient, and for which no standard therapy exists thus far. Patients suffering from AK are thus treated, out of necessity, with an off-label therapy, using drugs designed and indicated for other diseases/purposes. Here, we tested the capability of the off-label anti-amoebic drugs chlorhexidine (CH; 0.1%), dibromopropamidine diisethionate (DD; 0.1%), hexamidine diisethionate (HD; 0.1%), miltefosine (MF; 0.0065%), natamycin (NM; 5%), polyhexamethylene biguanide (PHMB; 0.02%), povidone iodine (PVPI; 1%), and propamidine isethionate (PD; 0.1%) to suppress trophozoite formation of Acantamoeba castellanii and Acanthamoeba hatchetti cysts on non-nutrient agar Escherichia coli plates. Of the eight off-label anti-amoebic drugs tested, only PVPI allowed for a complete suppression of trophozoite formation by drug-challenged cysts for all four Acanthamoeba isolates in all five biological replicates. Drugs such as NM, PD, and PHMB repeatedly suppressed trophozoite formation with some, but not all, tested Acanthamoeba isolates, while other drugs such as CH, DD, and MF failed to exert a relevant effect on the excystation capacities of the tested Acanthamoeba isolates in most, if not all, of our repetitions. Our findings suggest that pre-testing of the AK isolate with the non-nutrient agar E. coli plate assay against the anti-amoebic drug intended for treatment should be performed to confirm that the selected drug is cysticidal for the Acanthamoeba isolate.

Clinical presentations, genotypic diversity and phylogenetic analysis of Acanthamoeba species causing keratitis.

Introduction. Acanthamoeba keratitis is a sight-threatening corneal infection that is commonly reported among contact lens users and those suffering from corneal trauma. The prevalence of Acanthamoeba species or genotypes in causing keratitis infection is not well known.Aim. This study was conducted to identify and genotype Acanthamoeba isolates from keratitis patients, targeting the ribosomal nuclear subunit (Rns) region, and describe the associated clinical presentation and treatment outcome.Methodology. Thirty culture-confirmed patients with Acanthamoeba keratitis, identified in a tertiary eye care centre in South India during the period from December 2016 to December 2018, were included in this study. The data collected from patient records include demographic details, history of illness, mode of trauma, treatment history and follow-up status. The genotype and species were identified based on the Rns sequence and phylogenetic tree analysis.Results. Acanthamoeba culbertsoni was the most predominant keratitis-causing species, followed by Acanthamoeba quina, Acanthamoeba castellanii, Acanthamoeba healyi, Acanthamoeba hatchetti, Acanthamoeba polyphaga and Acanthamoeba stevensoni. Three major genotypes were identified (T4, T11 and T12), with the T4 genotype being the most predominant, with four subclusters, i.e. T4A, T4B, T4D and T4E. This is the first report on corneal infection by the A. stevensoni T11 genotype and the A. healyi T12 genotype. No significant correlation was observed between the clinical outcomes of corneal disease and the genotypes or species.Conclusion. Rns genotyping is very effective in identifying the Acanthamoeba species and genotype in keratitis. Genotyping of Acanthamoeba spp. will help to advance our understanding of genotype-specific pathogenesis and geographical distribution.

In Vitro Amoebicidal Activity of Titanium Dioxide/UV-A Combination Against Acanthamoeba.

To assess the amoebicidal effect of titanium dioxide (TiO2)/UV-A combination against Acanthamoeba sp trophozoites and cysts. The amoebicidal effect of the TiO2/UV-A combination was tested on trophozoites and cysts of clinical isolates of Acanthamoeba hatchetti and Acanthamoeba sp genotype T4, obtained from two severe cases of ulcerative keratitis. Samples of cultured Acanthamoeba were transferred to a 96-well plate. We tested the effect of sterile water (blank control), TiO2 alone, UV-A alone, TiO2 and additional UV-A exposure, chlorhexidine 0.02% alone, chlorhexidine 0.02% and TiO2, chlorhexidine and UV-A, chlorhexidine 0.02% and TiO2, and additional UV-A exposure. Cell viability assessment was done using the trypan blue dye exclusion method. The combination of TiO2 with UV-A demonstrated antitrophozoite and anticyst activity (P < 0.05). This in vitro study showed a synergistic effect of the association of chlorhexidine with TiO2 and UV-A on cysts (P < 0.001). Given the in vitro synergistic effectiveness of the association of chlorhexidine with TiO2 and UV-A against cysts, the treatment of Acanthamoeba keratitis could be improved by this new therapeutic approach.

Free-living protozoa in the gastrointestinal tract and feces of pigs: Exploration of an unknown world and towards a protocol for the recovery of free-living protozoa

Associations with free-living protozoa (FLP) have been implicated in the persistence of foodborne pathogenic bacteria in food-related environments. To date however no information is available on the presence of FLP in the gastrointestinal tract (GIT) of pigs, which represents an important reservoir for zoonotic foodborne bacteria and hence a potential location for associations with FLP. This is at least partly due to the lack of adequate protocols to recover FLP from intestinal content and feces. In the present study different protocols to recover FLP from the porcine GIT and feces were tested. The most effective protocols were then applied to explore the presence of live FLP in the pig GIT and feces. A filtration based protocol was identified as the most suitable method to recover viable FLP from the porcine GIT and feces. Cultivable FLP were recovered from different parts of the GIT, suggesting at least a transient presence of FLP in this habitat. Free-living amoebae species (Acanthamoeba spp., Hyperamoeba sp., Vannella sp., Vermamoeba vermiformis, hartmannellids and vahlkampfiids) but also ciliates (Colpoda sp. and Tetrahymena/Glaucoma lookalike) and flagellates (cercomonads, bodonids and glissomonads) were recovered and cultured from pig intestinal content. Acanthamoeba hatchetti and Filamoeba sinensis were isolated for the first time from pig intestinal content. Despite high gastric acidity, non-cyst forming amoeba species were also detected which suggests survival of their trophozoites in the animal GIT.

A clinical Acanthamoeba isolate harboring two distinct bacterial endosymbionts

Acanthamoebae feed on bacteria but are also frequent hosts of bacterial symbionts. Here, we describe the stable co-occurrence of two symbionts, one affiliated to the genus Parachlamydia and the other to the candidate genus Paracaedibacter (Alphaproteobacteria), within a clinical isolate of Acanthamoeba hatchetti genotype T4. We performed fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) to describe this symbiosis. Our study adds to other reports of simultaneous co-occurrence of two symbionts within one Acanthamoeba cell.

Occurrence and genetic characterisation of Acanthamoeba spp. from environmental and domestic water sources in Queen Elizabeth Protected Area, Uganda.

BackgroundAcanthamoeba is an emerging potentially pathogenic amoeba that has been receiving increasing attention worldwide as a reservoir and potential vector for the transmission of pathogenic bacteria. It is also associated with brain cell damage, keratitis and skin irritation in humans. Its effects are more severe in immunocompromised individuals. This study provides for the first time in Uganda, information on the prevalence and genotypes of Acanthamoeba in environmental and domestic (tap) water.MethodsA total of 324 environmental and 84 tap water samples were collected between November 2013 and September 2014. The samples were centrifuged, cultured (Non-Nutrient agar seeded with gram-negative bacteria) and observed under a microscope. After confirmation of Acanthamoeba, genomic DNA was extracted for PCR assays by chemical lysis and purification with phenol/chloroform/isoamyl alcohol. Samples that showed the strongest positive bands (400–600 bp) were subjected to cycle sequencing.ResultsAmong environmental and tap water samples, 107 (33 %) and 36 (42.9 %) tested positive for Acanthamoeba spp., respectively. Prevalence of Acanthamoeba from specific environmental locations was as follows; Kazinga channel banks (60.7 %), Fish landing sites (50 %), River Kyambura (39.6 %) and Kazinga mid channel (5.3 %). There was a significant difference (p = 0.001) in the prevalence of Acanthamoeba between sampling sites. The mean (Mean ± SE) occurrence of the organism was higher in Kazinga channel banks (3.44 ± 0.49) and Fish landing sites (3.08 ± 0.53). Correlation between in situ parameters and Acanthamoeba was insignificant except for the Dissolved Oxygen (mg/ML) which was negatively correlated (r = −0.231, p = 0.001) to Acanthamoeba. Six distinct partial Acanthamoeba T-genotype groups T1, T2, T4, T5, T6 and T11 were obtained. Ultimately, Acanthamoeba spp., Acanthamoeba hatchetti and Acanthamoeba polyphaga were isolated in the current study.ConclusionsThere was a high prevalence of Acanthamoeba in communal piped tap and environmental water used by communities, indicating poor environmental and domestic water quality.

Identification and quantification of the Acanthamoeba species and genotypes from reservoirs in Taiwan by molecular techniques

The occurrence of Acanthamoeba was investigated from 21 main reservoirs of Taiwan with 12 (57.1%) testing positive. Analysis of the 18S rRNA gene PCR product was performed in order to identify the Acanthamoeba isolates. Acanthamoeba spp. concentrations were determined according to TaqMan real-time qPCR. Acanthamoeba genotypes of all isolates were identified T4. The species were categorized to Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba hatchetti. The concentration of Acanthamoeba spp. in detected positive reservoir water samples was in the range of 3.0–1.8×103 cells/L. These results highlight the importance of Acanthamoeba in reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Miltefosine and polyhexamethylene biguanide: a new drug combination for the treatment of Acanthamoeba keratitis

In this study, a series of compounds - miltefosine, polyhexamethylene biguanide, chlorhexidine and propamidine isethionate - and combinations of the latter three agents with miltefosine were prepared and used in a rat model for the topical treatment of Acanthamoeba keratitis. The corneas of rats were infected with Acanthamoeba hatchetti. On the fifth day, all corneas were microscopically examined in order to determine the grade of infections. Nine groups were then prepared: miltefosine (65.12 μg/mL); chlorhexidine (0.02%); polyhexamethylene biguanide (0.02%), propamidine isethionate (0.1%), miltefosine plus chlorhexidine, miltefosine plus polyhexamethylene biguanide; miltefosine plus propamidine isethionate; infected control; and a non-infected control group. The treatment was continued for 28 days. After the treatment, the corneas were excised and used for Acanthamoeba culture to investigate the presence of Acanthamoeba growth. For the determination of cytotoxicity of the drugs on L929 cells, colorimetric assays were performed. The best treatment results were obtained from the polyhexamethylene biguanide plus miltefosine group; the ratio of fully recovered eyes was 28.4%. It was proven that the miltefosine-polyhexamethylene biguanide combination yielded the highest anti-acanthamoebal activity in that approximately 86% of the eyes were cleared from amoebae. The cytotoxicity values of the miltefosine and the control groups were compared with other groups and found to be statistically different (P < 0.05). This in vivo study demonstrates that a miltefosine-polyhexamethylene biguanide combination is highly effective for the treatment of Acanthamoeba keratitis.

Diversity and Seasonal Impact ofAcanthamoebaSpecies in a Subtropical Rivershed

This study evaluated the presence of Acanthamoeba species in the Puzih River watershed, which features typical subtropical monsoon climate and is located just above the Tropic of Cancer in Taiwan. The relationship between the seasonal and geographical distributions of Acanthamoeba species in this rivershed was also investigated. Acanthamoeba species were detected in water samples using the amoebal enrichment culture method and confirmed by PCR. A total of 136 water samples were included in this study, 16 (11.7%) of which contained Acanthamoeba species. Samples with the highest percentage of Acanthamoeba (32.4%) were obtained during the summer season, mainly from upstream areas. The identified species in the four seasons included Acanthamoeba palestinensis (T2), Acanthamoeba sp. IS2/T4 (T4), Acanthamoeba lenticulata (T5), Acanthamoeba hatchetti (T11), Acanthamoeba healyi (T12), and Acanthamoeba jacobsi (T15). The most frequently identified Acanthamoeba genotype was T4 (68.7%). Acanthamoeba genotype T4 is responsible for Acanthamoeba keratitis and should be considered for associated human health risk potential in the rivershed.

Cytotoxic effect of acriflavine against clinical isolates of Acanthamoeba spp

Acanthamoeba keratitis (AK) is a potentially devastating and sight-threatening infection of the cornea caused by the ubiquitous free-living amoebae, Acanthamoeba species. Its eradication is difficult because the amoebas encyst, making it highly resistant to anti-amoebic drugs. Acriflavine neutral (ACF) has been used for treatment of microbial infections for humans and fishes. The aim of our study was to evaluate the time-dependent cytotoxicities of ACF against Acanthamoeba spp. Trophozoites and cysts of three different strains (strain PAT06 Acanthamoeba castellanii, strain 2HH Acanthamoeba hatchetti, and strain 11DS A. hatchetti) of Acanthamoeba spp. were tested. All strains had been isolated from patients suffering from a severe AK. The effects of the ACF with the concentrations ranging from 15 to 500 mg mL(-1) on the cytotoxicity of Acanthamoeba strains were examined. ACF showed a time- and dose-dependent amebicidal action on the trophozoites and cysts. Pat06 (A. castellanii) was the most resistant, while strain 11DS (A. hatchetti) was the most sensitive. As a result, ACF could be concluded as a new agent for the treatment of Acanthamoeba infections. On the other hand, it still needs to be further evaluated by in vivo test systems to confirm the efficiency of its biological effect.

Miltefosine and polyhexamethylene biguanide, a new drug combination for the treatment of Acanthamoeba keratitis. Results from in‐vivo toxicological and efficacy studies

AbstractPurpose Miltefosine (MLT) and combinations of Miltefosine and polyhexamethylene biguanide (PHMB), chlorhexidine (CHX), and propamidine isethionate (PI), respectively, have been tested in a rat efficacy model for treatment of Acanthamoeba keratitis. In a second step, the most promising treatment regimens from efficacy studies have been tested for local tolerance in a fully GLP‐compliant toxicological study in pigmented rabbits.Methods The cornea of rats were infected with Acanthamoeba hatchetti. Cornea infections were graded microscopically. Nine groups were treated with varous treatment regimens for 28 days (8 times per day during the first week, and 3 times per day for the last three weeks). Efficacy of treatment was examined by determination of the degree of infection and culturing of excised eyes after the end of the treatment period. On day 28 the eyes were inspected under the microscope, re‐graded and were then compered with the grades before starting the therapy.Results Best treatment results were obtained from PHMB‐MLT group. The ratio of fully recovered eyes was 28.4%. The highets therapeutic activity was yielded by the combination MLT‐PHMB (86%), followed by MLT, CHX, or PI (72%) and the combination MLT–PI (70%. The fully GLP‐compliant study in pigmented rabbits (28 days, 8 treatments per day) showed excellent local tolerance.Conclusion Thus the combination of Miltefosine and PHMB can be seen as a highly effective and save option for treatment of AK.

Efficacy of miltefosine for topical treatment of Acanthamoeba keratitis in Syrian hamsters

Acanthamoeba keratitis is a painful corneal infection and difficult to treat because no sufficiently efficient drug has yet been available. The aim of the study therefore was to assess the therapeutic potential of miltefosine on Acanthamoeba keratitis-infected hamster eyes. The cornea of hamsters were infected with Acanthamoeba hatchetti, a human corneal isolate. On the fifth day, all the cornea were microscopically examined in order to determine the degree of infections (G, from 0 to 3). Four groups were then prepared: miltefosine (160 μM); 0.1% propamidine isetionate plus 0.02% polyhexnide; infected control (0.05% ethanol in PBS) and a non-infected control (0.05% ethanol in PBS) groups. The treatment was continued for 28 days. After the treatment, the cornea were excised and used for Acanthamoeba culture to investigate the presence of Acanthamoeba growth. Miltefosine treatment yielded much higher cure scores than propamidine isetionate plus polyhexanide. On the last day of treatment, 85% of the miltefosine-treated eyes were graded as G0; no changes were observed in the uninfected control group eyes; G3 eyes showed only a partial improvement. Furthermore, no Acanthamoeba cells could be recovered from the miltefosine-treated eye samples. Miltefosine appeared to hold necessary therapeutic properties for the treatment of Acanthamoeba keratitis.

Adaptation of amoebae to cooling tower biocides

Adaptation of amoebae to four cooling tower Biocides, which included a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds (TBT/QAC), another QAC alone, and an isothiazolin derivative, was studied. Previously we found that amoebae isolated from waters of cooling towers were more resistant to cooling tower biocides than amoebae from other habitats. Acanthamoeba hatchetti and Cochliopodium bilimbosum, obtained from American Type Culture Collection and used in the previous studies, were tested to determine whether they could adapt to cooling tower Biocides. A. hatchetti was preexposed to subinhibitory concentrations of the four Biocides for 72h, after which they were tested for their resistance to the same and other biocides. C. bilimbosum was exposed to only two biocides, as exposure to the other two was lethal after 72 h. Preexposure to the subinhibitory concentrations of the Biocides increased the resistance of the amoebae, as indicated by a significant increase in the minimum inhibitory concentration (up to 30-fold). In addition, cross-resistance was also observed, i.e., exposure to one biocide caused resistance to other biocides. These results show that amoebae can adapt to biocides in a short time. The phenomenon of cross-resistance indicates that regularly alternating biocides, as is done to control microbial growth in cooling towers, may not be effective in keeping amoeba populations in check. On the contrary, exposure to one biocide may boost the amoebae's resistance to a second biocide before the second biocide is used in the cooling tower. Since amoebae may harbor Legionella, or alone cause human diseases, these results may be important in designing effective strategies for controlling pathogens in cooling towers.

Stimulatory effect of cooling tower biocides on amoebae

Two species of amoebae were isolated from the cooling tower of an air-conditioning system and examined for effects of exposure to four cooling tower biocides, a thiocarbamate compound, tributyltin neodecanoate mixed with quaternary ammonium compounds, another quaternary ammonium compound alone, and an isothiazolin derivative. The amoebae isolated were Acanthamoeba hatchetti and a Cochliopodium species. Two other amoeba cultures, an A. hatchetti culture and Cochliopodium bilimbosum, were obtained from the American Type Culture Collection (ATCC) and were also tested. The cooling tower isolates were more resistant to most of the biocides than the ATCC isolates were. The isothiazolin derivative was the least inhibitory to all four amoeba isolates, and tributyltin neodecanoate mixed with quaternary ammonium compounds was the most inhibitory to three of the four isolates. After exposure to lower concentrations of the biocides, including for one strain the manufacturer's recommended concentration of one biocide, the cooling tower amoeba populations increased significantly compared with unexposed controls, whereas the ATCC isolates were not stimulated at any of the concentrations tested. In some cases, concentrations which stimulated cooling tower amoebae inhibited the growth of the ATCC isolates. These results suggest that cooling tower amoebae may adapt to biocides, underscoring the need to use freshly isolated cooling tower organisms rather than organisms from culture collections for testing the efficacy of such biocides. The stimulatory effect of biocides on amoeba populations is an alarming observation, since these organisms may be reservoirs for legionellae. Biocides used to control microbial growth may actually enhance populations of host organisms for pathogenic bacteria.

Incubation of water samples containing amoebae improves detection of legionellae by the culture method

Some protozoans isolated from aquatic habitats, including domestic water supplies, can support the intracellular replication of autochthonous legionellae in vitro. We studied the effect of incubating water samples containing amoebae on the sensitivity of culture for legionellae. Samples collected during investigations of legionellosis epidemics and shown by conventional culture procedures to contain amoebae, but not legionellae, were incubated at 35 degrees C and replated. Legionellae were recovered from 59 of 144 such samples. Species isolated included L. pneumophila, L. anisa, L. bozemanii, L. gormanii, L. micdadei, L. rubrilucens, L. sainthelensi, L. steigerwaltii, and an unnamed species. Acanthamoeba polyphaga, Acanthamoeba hatchetti, a Rosculus sp., Hartmannella vermiformis, and Vahlkampfia spp. were among the autochthonous amoebae identified. Legionellae were recovered by this procedure from only 3 of 63 samples that were negative for amoebae by conventional culture procedures. These results show that water samples negative for legionellae, but positive for amoebae, by standard culture techniques should be incubated and replated to maximize the sensitivity of culture for legionellae.

Pathogenic Amoebas from Brackish and Ocean Sediments, with a Description of Acanthamoeba hatchetti , n. sp.

Acanthamoeba culbertsoni was isolated from a sewage-spoil dump site near Ambrose Light, New York Bight. A second species, Acanthamoeba hatchetti, n. sp., was isolated from Brewerton Channel, Baltimore Harbor, Maryland. Both species killed laboratory mice after infection by the intranasal route.