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.     

<Emphasis Type="Italic">Acanthamoeba</Emphasis> T4 and T15 genotypes associated with keratitis infections in Italy

Thus far there is little data available concerning Acanthamoeba associated amoebic keratitis (AK) from Italy. In order to understand the incidence of Acanthamoeba in patients with ocular infections and to characterize the isolates at the molecular level, ocular specimens and contact lenses or lens case solutions from 140 patients were analysed by culture and by an 18S rRNA (Rns) gene-based PCR method. Nineteen (13.6%) patients showed Acanthamoeba culture positive samples. Eleven out of the 14 genetically characterized isolates were assigned to the T4 genotype. Three isolates, two of them from patients with keratitis responding to specific anti-Acanthamoeba therapy, were identified as belonging to the T15 genotype. This finding represents the first association between the T15 genotype and human amoebic keratitis. PCR amplification of the 18S ribosomal DNA proved to be a sensitive method, potentially able to detect Acanthamoeba without the need of long culture incubation, and thus considerably useful for clinical applications.     

Comparison of PCR,microscopic examination and culture for the early diagnosis and characterization of <Emphasis Type="Italic">Acanthamoeba</Emphasis> isolates from ocular infections

In the study presented here, PCR, microscopic examination and culture of corneal samples were compared as methods of confirming the clinical diagnosis of Acanthamoeba keratitis, a serious ocular infection that is difficult to diagnose and threatens eyesight. The three methods were applied to isolates obtained from 513 patients with clinical signs or risk factors suggesting Acanthamoeba infection. Acanthamoeba keratitis was diagnosed in 12 of these patients. Combined PCR assays were more sensitive (94%) than either microscopic examination (33%) or culture (7%). The Acanthamoeba isolates were characterized using DNA sequence analysis of the nuclear small-subunit rRNA gene, and T4 was the predominant genotype found.     

First report of an Acanthamoeba genotype T13 isolate as etiological agent of a keratitis in humans

Several strains of free-living amoebae (FLA) belonging to the genus Acanthamoeba are able to cause a painful sight-threatening disease of the cornea designated as Acanthamoeba keratitis (AK). In this case report, a 22-year-old woman, wearer of soft contact lenses, was treated after the initial examination, and follow-up laboratory results led to the diagnosis of Acanthamoeba keratitis. The patient recovered under the targeted therapy, demonstrating that the acanthamoebae were the etiological agents of the keratitis in this case. The acanthamoebae belonged morphologically to group II. Genotyping of the causative Acanthamoeba strain based on sequences of the PCR amplimer ASA.S1 amplified from 18S ribosomal DNA by using the genus-specific primers JDP1 and JDP2 followed. The phylogenetic comparison of ASA.S1 confirmed that the isolated Acanthamoeba strain is closely related to genotype T13 supported by pairwise sequence identities of 97.1–98.0 % and bootstrap support of 980 replicates with reference sequences of genotype T13. These results regarding the Acanthamoeba keratitis-causing isolate KaBo expands the number of known pathogenic genotypes to 12. To our knowledge, this is the first report of a T13 Acanthamoeba genotype being associated with keratitis in humans.     

Evaluation of the effectiveness of tea tree oil in treatment of <Emphasis Type="Italic">Acanthamoeba</Emphasis> infection

Eye diseases caused by amoebae from the genus Acanthamoeba are usually chronic and severe, and their treatment is prolonged and not very effective. The difficulties associated with therapy have led to attempts at finding alternative treatment methods. Particularly popular is searching for cures among drugs made of plants. However, no substances with total efficacy in treating Acanthamoeba keratitis have been identified.Results of our semi in vivo studies of tea tree oil simulating eyeball infection demonstrated 100% effectiveness in the case of both trophozoites and cysts of amoebae from the genus Acanthamoeba. The action of tea tree oil indicates that this is the first substance with a potential ability to quickly and effectively remove the amoebae from the eye. Tea tree oil has the ability to penetrate tissues, which allows it to destroy amoebae in both the shallow and deep layers of the cornea. The present research into the use of tea tree oil in the therapy of Acanthamoeba infection is the first study of this type in parasitology. It offers tremendous potential for effective treatment of Acanthamoeba keratitis and other diseases caused by these protozoa.     

A Rabbit Model of Acanthamoeba Keratitis That Better Reflects the Natural Human Infection

Acanthamoeba species are ubiquitous, free‐living protozoa that can invade the cornea and result in Acanthamoeba keratitis (AK), a painful progressive sight‐threatening corneal disease. Disease progression in current animal models is too rapid to mimic AK in humans accurately. This study provides a novel method for establishing AK in rabbits and compared it with the conventional method with regard to pathogenesis and immune response in humans. The New Zealand white rabbits were randomly divided into two experimental groups (Groups A and B). Rabbits in the Group A (n = 14) received intrastromal injections of 1 × 10⁴/100 µL Acanthamoeba healyi trophozoites (conventional AK model). The Group B animals (n = 14) received microinjections of 1 × 10⁴/10 µL A. healyi trophozoites between the corneal epithelium and Bowman's layer, anterior to the corneal stroma (novel AK model). In addition, two rabbits were left untreated as normal controls. AK in the treated rabbits was evaluated clinically, histopathologically, and immunologically for 35 days. AK was successfully established in both the conventional and novel model groups. Compared with the Group A, AK in the Group B displayed an efficient immune response with less severe pathology. Moreover, the self‐limiting but chronic nature of the infection in the Group B was strikingly similar to that of AK in humans. The novel animal model for AK described here more closely simulates the pathogenesis and immune response of Acanthamoeba corneal infection in humans than the animal models currently in use. Anat Rec, 298:1509–1517, 2015. © 2015 Wiley Periodicals, Inc.     

Comparison of a novel semi-nested polymerase chain reaction (PCR) with a uniplex PCR for the detection of <Emphasis Type="BoldItalic">Acanthamoeba</Emphasis> genome in corneal scrapings

A semi-nested polymerase chain reaction (snPCR) was developed to improve the sensitivity of detection of Acanthamoeba sp. genome from corneal scrapings of Acanthamoeba keratitis patients. The snPCR was developed using a laboratory designed inner forward primer targeting the 450-bp product of the 18s rRNA-gene-based PCR. The snPCR was optimized using 11 Acanthamoeba sp. culture isolates and further applied onto 35 corneal scrapings from keratitis patients. The sensitivity and specificity of the snPCR was compared against conventional methods (smear and/or culture-gold standard) and the uniplex PCR described by Schroeder et al. Eleven out of the 35 corneal scrapings were positive by the gold standard and snPCR, whereas only 3 of these 11 were positive by the uniplex PCR. The clinical sensitivity and specificity of the snPCR was 100% when compared with the gold standard. DNA sequencing was performed on first round amplicons of four culture isolates and one specimen, and all of them were identified as genus Acanthamoeba when compared with the GenBank database sequences. Application of snPCR on the 11 culture isolates yielded amplicons ranging 120–160 bp in size, indicating sequence variation among the different culture isolates. The clinical sensitivity of snPCR was higher than the conventional methods and the uniplex PCR reported earlier.     

<Emphasis Type="Italic">Acanthamoeba</Emphasis> keratitis due to <Emphasis Type="Italic">Acanthamoeba</Emphasis> genotype T4 in a non-contact-lens wearer in Turkey

An otherwise healthy 22-year-old man presented with Acanthamoeba keratitis (AK) in the right eye. He was not a contact lens wearer and had no history of corneal trauma. The Acanthamoeba strain isolated from a corneal scraping was identified as morphological group II and genotype T4. Three more Acanthamoeba strains isolated from sites of possible human contact with acanthamoebae in the same geographical region, including a lens storage case, tap water and soil, were subjected to morphological and molecular biological identification. Whereas the strain from tap water also exhibited genotype T4, the two other isolates were identified as morphological group I and genotype T9. To the best of our knowledge, this is the first study identifying an AK-causing Acanthamoeba strain in Turkey and the first isolation of genotype T9 in this country.The authors declare that all experiments performed comply with the current laws of Turkey and Austria.     

Evaluation of Three Different Methods to Establish Animal Models of Acanthamoeba Keratitis

Purpose

To produce animal models of Acanthamoeba keratitis and to evaluate the advantages and adaptation range of each of the three methods employed.

Materials and Methods

Mice and Wistar rats in three groups of 15 rats and 15 mice each were used to establish the models. Right corneas in group A were scratched and challenged with Acanthamoeba. Those in group B were scratched and covered with contact lenses incubated with Acanthamoeba. Those in group C received an intrastromal injection of Acanthamoeba. Five rats and 5 mice in each group were used for histopathological investigations and the other 10 in each group were used for clinical evaluation. The models were evaluated by slit lamp examination, microscopic examination and culture of corneal scrapings, HE staining of corneal sections, and pathological scoring of the infections.

Results

Four rats and 6 mice in group A, 7 rats and 8 mice in group B, and 10 rats and 10 mice in group C developed typical Acanthamoeba keratitis.

Conclusion

Corneal scratching alone has the lowest infection rate, while scratching and then covering with contaminated contact lenses has a moderate rate of infection and most closely mimics what happens in most human infections. Intrastromal injection of Acanthamoeba gives a much higher infection rate and more severe Acanthamoeba keratitis.     

The use of high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy in the clinical diagnosis of Acanthamoeba

Acanthamoeba are opportunistic protozoan pathogens that can produce sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba spp. followed by aggressive treatment regimen. In this study, we tested the use of high-resolution ¹H NMR spectroscopy in the clinical diagnosis of Acanthamoeba. Using NMR spectroscopy combined with Pattern Recognition Analysis (PRA), we analysed variations in the biochemical ‘fingerprint’ of invasive and non-invasive Acanthamoeba, its closely related genus, Balamuthia mandrillaris, neuropathogenic Escherichia coli K1 strain E44, a laboratory strain of E. coli K-12, HB101, mammalian cells including human brain microvascular endothelial cells and monkey kidney cells. The findings revealed significant variations in the metabolites of amoebae, mammalian cells and bacteria. Interestingly, ¹H NMR spectra provided distinct biochemical profiles of clinical and non-clinical Acanthamoeba isolates highlighting the potential of ¹H NMR spectroscopy combined with PRA for the development of a novel diagnostic test that could rapidly identify pathogenic Acanthamoeba isolates with high sensitivity and specificity.     

Characterisation and differentiation of pathogenic and non-pathogenic<Emphasis Type="Italic"> Acanthamoeba</Emphasis> strains by their protein and antigen profiles

Free-living amoebae of the genus Acanthamoeba are the causative agents of Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis. Acanthamoebae occur ubiquitously in the environment and are thus a constant cause of antigenic stimulation. In a previous study we have shown that compared to control sera, AK patients exhibit markedly lower immunoreactivities to whole cell antigen of Acanthamoeba spp. As the pathogenicity of acanthamoebae primarily relies on the excretion of proteins, it was the aim of the present study to investigate the immunoreactivity of metabolic antigen from different Acanthamoeba strains of varying pathogenicity. Three Acanthamoeba strains, one highly pathogenic, one non-pathogenic but thermophilic and one non-thermophilic non-pathogenic, were used for antigen extraction. The antigen was harvested before and after contact with human cells and all strains were tested with AK sera and with sera from healthy individuals. It was shown that the somatic protein profiles of the Acanthamoeba strains correlated to the morphological groups, and that within morphological group II—the group associated with AK—the profiles of the metabolic antigens correlated to strain pathogenicity. Moreover, it was shown that the control sera showed markedly higher immunoreactivities than the sera of the AK patients and that this immunoreactivity was generally higher to the non-pathogenic strains than to the pathogenic strain. Altogether our results once again raise the question of whether there is an immunological predisposition in AK. To our knowledge this is the first study on the immunoreactivity of metabolic antigen of acanthamoebae.     

Flagellin‐induced expression of CXCL10 mediates direct fungal killing and recruitment of NK cells to the cornea in response to Candida albicans infection

We previously showed that topical flagellin induces profound mucosal innate protection in the cornea against microbial infection, a response involving multiple genes and cell types. In this study, we used a Candida albicans (CA)‐C57BL/6 mouse keratitis model to delineate the contribution of CXCL10‐ and CXCR3‐expressing cells in flagellin‐induced protection. Flagellin pretreatment markedly enhanced CXCL10 expression at 6 h post CA infection (hpi), but significantly dampened CXCL10 expression at 24 hpi. At the cellular level, CXCL10 was expressed in the epithelia at 6 hpi in flagellin‐pretreated corneas, and concentrated at lesion sites 24 hpi. CXCR3‐expressing cells were detected in great numbers at 24 hpi, organized within clusters at the lesion sites in CA‐infected corneas. CXCL10 or CXCR3 neutralization increased keratitis severity and dampened flagellin‐induced protection. CXCR3‐positive cells were identified as NK cells, the depletion of which resulted in severe CA keratitis. Contributions from NK T‐cells were excluded by finding no change in flagellin‐induced protection in Rag1 KO mice. Recombinant CXCL10 inhibited CA growth in vitro and accelerated fungal clearance and inflammation resolution in vivo. Taken together, our data indicate that epithelium‐expressed CXCL10 plays a critical role in fungal clearance and that CXCR3‐expressing NK cells contribute to CA eradication in mouse corneas.     

Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of Acanthamoeba. The sensitivity of the LAMP assay was tested using different copies of positive DNA. The specificity of the assay was tested using DNA extracted from Acanthamoeba, Pseudomonas aeruginosa, Candida albicans, herpes simplex virus-1 and human corneal epithelial cells. Its effectiveness was evaluated and compared with culture, corneal smear examination and real-time PCR in corneal samples from mice with Acanthamoeba keratitis. We also tested three corneal samples from patients with suspected Acanthamoeba or fungal infection using LAMP. Loop-mediated isothermal amplification was confirmed to be very sensitive, with the lowest detection limit being ten copies/tube of Acanthamoeba DNA. The LAMP primers only amplified Acanthamoeba DNA. During the development of Acanthamoeba keratitis in mice, almost all of the positive rates of LAMP at each time post-infection were higher than those of culture or corneal smear examination. The total positive rate of LAMP was significantly higher than those of culture and corneal smear examination (p <0.05), whereas the sensitivities of LAMP and real-time PCR were comparable. However, the trends of positive change in these different test methods were generally similar. Of the three clinical corneal specimens, two with suspected Acanthamoeba keratitis tested positive for Acanthamoeba using LAMP along with culture or corneal smear examination, whereas the other suspected fungal keratitis tested negative. The LAMP assay is a simple, rapid, highly specific and sensitive method for the diagnosis of keratitis caused by Acanthamoeba.     

In vitro effectiveness of Thymus sipyleus subsp. sipyleus var. sipyleus on Acanthamoeba castellanii and its cytotoxic potential on corneal cells

Acanthamoeba species are an important cause of microbial keratitis that may cause severe ocular inflammation and visual loss. Acanthamoeba keratitis is difficult to treat, without total efficacy in some patients because of cysts which is less susceptible than trophozoites to the usual treatments. We investigated here the in vitro amoebicidal activity of methanolic extract of Thymus sipyleus subsp. sipyleus var. sipyleus from Turkish flora against Acanthamoeba castellanii and also its cytotoxicity on corneal cells in vitro. Extract was evaluated for its amoebicidal activity using an inverted light microscope. The effect of the polar extract with the concentrations ranging from 1.0 to 32.0 mg/ml on the proliferation of A. castellanii trophozoites and cysts were examined in vitro. For the determination of cytotoxicity of the extract on corneal cells, agar diffusion tests were performed. According to results obtained from the tests, the extract evaluated here showed remarkable amoebicidal effect on A. castellanii. In the case of the cytotoxic activities, it showed no cytotoxicity for corneal cells in the concentration of 32 mg/ml. As a result, polar subfraction of the methanolic extract of Thymus sipyleus subsp. sipyleus var. sipyleus could be concluded as a new natural 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.     

Phylogenetic evidence for a new genotype of Acanthamoeba (Amoebozoa, Acanthamoebida)

Acanthamoeba are widespread free-living amoebae, able to cause infection in animals, with keratitis and granulomatous encephalitis as major diseases in humans. Recent developments in the subgenus classification are based on the determination of the nucleotide sequence of the 18S rDNA. By this mean, Acanthamoeba have been clustered into 15 sequence types or genotypes, called T1 to T15. In this study, we analysed near full 18S rDNA of an Acanthamoeba recovered from an environmental sample and various unidentified Acanthamoeba sequences retrieved from GenBank. We provided phylogenetic evidence for a new genotype, which we proposed to name T16.     

Early diagnosis of amoebic keratitis due to a mixed infection with <Emphasis Type="Italic">Acanthamoeba</Emphasis> and <Emphasis Type="Italic">Hartmannella</Emphasis>

A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a soft contact lens wearer. Early diagnosis was achieved by polymerase chain reaction and culture. The pathogenic potential of the isolated amoebae was proven using cytotoxicity assays. The reported case underlines the difficulties in identifying a corneal amoebic infection. In our case, the early diagnosis of a mixed infection allowed a proper antiamoebic treatment in an early stage of infection. This may have been the reason of a successful outcome after therapy.     

Amebicidal activity of plant extracts from Southeast Asia on Acanthamoeba spp.

The effect of 100 polar and 100 nonpolar plant extract materials obtained from Southeast Asia were evaluated for amebicidal activity in vitro against three species of Acanthamoeba.A. culbertsoni, A. castellanii, and A. polyphaga, the causative agents of granulomatous amebic encephalitis and amebic keratitis, were studied in vitro to determine whether the plant extracts exhibited amebicidal activity or induced encystment of the amebae. Of the 200 plant extracts tested, extracts obtained from three plants (Ipomoea sp., Kaempferia galanga, and Cananga odorata) were amebicidal for all three species of Acanthamoeba and a fourth extract prepared from Gastrochilus panduratum was lytic for A. polyphaga and growth-inhibitory for A. castellanii and A. culbertsoni. Three plant extracts induced encystment of all three species of Acanthamoeba. Select plant extracts were tested as well for tumoricidal activity against B103 neuroblastoma cells. Some plant extracts that exhibited tumoricidal activity for B103 cells were not amebicidal for Acanthamoeba spp. Additionally, the polar and nonpolar extracts that exhibited amebicidal activity were also tested for activity against primary murine peritoneal macrophage cultures. Plant extracts that demonstrated tumoricidal or amebicidal activity were not lytic for normal macrophage cultures. Received: 23 February 1998 / Accepted: 18 May 1998     

Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis

Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.     

Effect of combined chlorhexidine gluconate and neosporin on experimental keratitis with two pathogenic strains of Acanthamoeba

Acanthamoeba keratitis (AK) is a painful, sight-threatening, and difficult-to-treat corneal infection caused by the ubiquitous free-living amoebae Acanthamoeba species. The aim of the present study was to compare the severity of keratitis, caused by Acanthamoeba hatchetii and Acanthamoeba castellanii infections, and to assess the therapeutic effects of combined chlorhexidine (CHX) and Neosporin? treatment in rats. The rats were first divided into two groups, in which the eyes of the animals were infected with A. hatchetii or A. castellanii trophozoites. On day 5, all corneas were examined in order to determine the degree of infection (grade 0 to 3), and animals were divided into two new groups, treatment and infected control groups. The treatment was continued for 28 days, followed by excision and histological evaluation of the corneas. In conclusion, the clinical picture progressed more rapidly and severely in eyes infected by A. castellanii, while it was non-invasive and slower to progress with A. hatchetii. Moreover, eyes infected by A. hatchetii responded quicker and more positively to therapy, consistent with its clinical course, while a longer recovery was seen with A. castellanii. Histological examinations revealed the presence of A. castellanii and A. hatchetii trophozoites in the stroma of eyes of the treatment and control groups. As a result, our findings suggest that a combination of Neosporin with lower doses of CHX may be beneficial to treat patients with early diagnosis of AK.     

Acanthamoeba keratitis in a mouse model using a novel approach

ContextAcanthamoeba is increasingly implicated in causing keratitis in patients wearing contact lens or ocular trauma and has a poor prognosis. Establishment of an animal model is critical to study the disease pathology, pathogenesis and to evaluate anti-amoebic drugs. Some studies have used contact lenses to establish Acanthamoeba keratitis (AK) in a mouse model, which is expensive and not very successful as lenses get dislodged.ObjectiveTo assess the feasibility of using parafilm (Bemis Company Inc., USA) as an alternative to contact lens for the establishment of AK in the mouse model.MethodsThirty-six Balb/c mice in three groups of six mice each for two strains of Acanthamoeba were used to induce AK. Three experimental approaches used were; i) Acanthamoeba impregnated contact lens, ii) Acanthamoeba impregnated parafilm and iii) scratching followed by inoculation of Acanthamoeba suspension. In all three models, tarsorrhaphy was performed. Infection was evaluated by clinical examination and also through microscopic examination of corneal scrapings and corneal sections.ResultsAK model was successfully established with parafilm whereas only one mouse developed AK with the use of contact lens and none with scratching and Acanthamoeba inoculation.ConclusionThe use of parafilm is convenient, reliable and cheaper and can be considered an alternative to contact lenses to induce AK in a mouse model.