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.     

Granulomatous inflammation in Acanthamoeba keratitis: an immunohistochemical study of five cases and review of literature

Purpose: Acanthamoeba keratitis usually presents as a necrotizing stromal inflammation. We report a rare presentation of granulomatous inflammation in Acanthamoeba keratitis Methods: Retrospective clinico-pathologic case series. Results: Five corneal tissues (3 corneal buttons, 2-eviscerated contents) from patients suffering from severe Acanthamoeba keratitis not responding to anti-Acanthamoeba treatment, revealed a florid granulomotous inflammation with multinucleated giant cells in the posterior stroma and around Descemet’s membrane. Phagocytosed parasites were noted within the giant cells. Vascularization of the corneal stroma was noted in two cases. Immunophenotyping revealed a predominance of T lymphocytes and macrophages. Clinically, four of five cases had shown features of limbal and scleral involvement. Conclusion: Granulomatous inflammation in the posterior corneal stroma, is not an uncommon finding in Acanthamoeba keratitis and could possibly be immune-mediated, contributing to persistence and progression of disease. Clinical Relevance: Presence of granulomatous inflammation in Acanthamoeba keratitis, in most cases is associated with limbal and scleral involvement and therefore could be considered as one of the poor prognostic markers. Further studies are required to ascertain the specific clinical features and appropriate management strategies in these cases.     

Stenotrophomonas maltophilia and Vermamoeba vermiformis relationships: Bacterial multiplication and protection in amoebal-derived structures

Stenotrophomonas maltophilia, a bacteria involved in healthcare-associated infections, can be found in hospital water systems. Other microorganisms, such as Free Living amoebae (FLA), are also at times recovered in the same environment. Amongst these protozoa, many authors have reported the presence of Vermamoeba vermiformis. We show here that this amoeba enhances S. maltophilia growth and harbors the bacteria in amoebal-derived structures after 28 days in harsh conditions. These results highlight the fact that particular attention should be paid to the presence of FLA in hospital water systems, because of their potential implication in survival and growth of pathogenic bacterial species.     

Free living amoebae in water sources of critical units in a tertiary care hospital in India

Background: Isolation of free-living amoebae (FLA) is reported sparsely from water taps, ventilators, air conditioners, haemodialysis units and dental irrigation systems of hospitals worldwide. Their prevalence in hospital environment especially in wards having immunocompromised patients may pose a risk to this group of susceptible population as they may cause disease themselves or may carry pathogens inside them. No study from India has performed such surveillance. Objective: To evaluate extent of FLA contamination in water sources of bone marrow transplant (BMT) intensive care unit (ICU), transplant ICU, haemodialysis unit and high dependency unit in a tertiary care hospital in India. Materials and Methods: A total of hundred samples including fifty each of tap water samples and swabs from mouth of taps used for drinking, bathing and hand washing purposes in these units were collected according to standard procedure. Samples were inoculated onto non-nutrient agar plates at room temperature followed by morphological confirmation. Molecular identification including polymerase chain reaction (PCR) and sequencing was performed in culture positive samples. Results: Four tap water samples and ten swab samples showed growth of trophozoites and cyst formation. Morphologically, four amoebae resembled Acanthamoeba spp. which was further confirmed by PCR and sequencing showed them to be of T3 and T4 genotypes. Conclusion: The presence of these FLA in hospital water sources emphasises the urgent need of implementing effective preventive measures. Further studies are required to estimate the true prevalence of FLA in Indian hospitals by taking larger number of samples.     

Use of different stains for microscopic evaluation for the diagnosis of Pythium keratitis

PurposeTo evaluate the efficacy of various staining techniques for detection of Pythium in keratitis cases.MethodsData of nineteen consecutive culture-positive cases of Pythium keratitis were retrospectively analysed. Corneal scrapings and corneal buttons (in the cases which underwent therapeutic penetrating keratoplasty [TPK]) were sent for microbiological and histopathological examination. The direct smears were stained with Potassium hydroxide and calcofluor white (KOH ?+ ?CFW), Gram and Iodine–Potassium Iodide–Sulphuric Acid (IKI–H₂SO₄) stains. The corneal buttons were stained with Gomori's Methanamine Silver (GMS), Periodic Acid-Schiff (PAS) and Iodine–Potassium Iodide–Sulphuric Acid (IKI–H₂SO₄) stains. The positivity of various stains in detecting Pythium was studied.ResultsGram and KOH ?+ ?CFW staining from smear was done in 16 out of 19 (84.2%) cases. KOH ?+ ?CFW and Gram stains were suggestive of Pythium in 10 (62.5%) and 7 (43.8%) cases, respectively. IKI–H₂SO₄ staining in scraping samples was positive for Pythium in all the 4 (100%) cases in which it was performed. Half corneal buttons were positive for Pythium with IKI–H₂SO₄ stain as well as GMS stain in all the 18 cases that underwent TPK (100%). PAS stain showed weak to faint pink staining of Pythium filaments in 7 out of 18 cases (38.9%).ConclusionIKI–H₂SO₄ stain followed by KOH ?+ ?CFW stain detects Pythium filaments most accurately in corneal scraping samples from keratitis patients, although the differences were not statistically significant. The positivity of the stains depends on astute observation by an experienced ocular microbiologist and pathologist.     

Evaluation of Loop-mediated Isothermal Amplification Assay for Rapid Diagnosis of Acanthamoeba Keratitis

Background: The clinical features of Acanthamoeba keratitis (AK) are non-specific and closely resemble bacterial, viral and fungal keratitis. Materials and Methods: We compared loop-mediated isothermal amplification (LAMP) with microscopy, non-nutrient agar (NNA) culture and polymerase chain reaction (PCR) in clinical suspects of AK. Results: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis), 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%–92.7%), and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%–100%). The limit of detection of Acanthamoeba DNA was 1 pg/μl by both LAMP and PCR. Conclusion: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2–3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings     

Identification of Acanthamoeba in bronchoalveolar lavage specimens.

Acanthamoeba species infect humans occasionally and act as opportunistic pathogens in immunocompromised individuals. This study demonstrates the application of cytocentrifugation as an aid to identification of Acanthamoebae. In addition, certain staining procedures clearly optimized visualization of characteristic amoebic features. This was demonstrated by adding amoebae from laboratory cultures to bronchoalveolar lavage specimens. In preparations stained by the Papanicolaou, trichrome, or hematoxylin-eosin (H&E) procedures, the discrete deeper staining nucleolus was the most distinctive feature. The vacuolated cytoplasm also aided in the identification of amoebae. These features were less apparent and often distorted following staining of Acanthamoeba species with the Hema III and Giemsa procedures.     

Acanthamoeba keratitis: about the first two Tunisian cases

Acanthamoeba keratitis is a rare but severe corneal infection which, despite improvements in diagnosis and treatment, still culminates in prolonged morbidity and significant loss of visual acuity. We present the case report of the first identification of Acanthamoeba as a causative agent of keratitis in Tunisia. Case no 1: A 20-year-old girl, nearsighted corrected with soft contact lenses, suffering from a deep corneal inflammation and poor visual acuity The ophthalmological examination showed bilateral dendritiform epithelial keratitis. The illness did not respond to topical and general antibiotic treatment and developed bilateral corneal abscess. Microscopic examination and culture of samples from cornea scraping revealed the presence of trophozoit and cysts of Acanthamoeba associated with Fusarium oxysporum. As the treatment with local Ketoconazol and antibiotherapy didn't show any result, two transfixiant keratoplasty were carried out and treatment by Désomédine, PHMB (polyhexamethylene biguanide) and Voriconazol was started. After two months, the patient felt better, vision was also improved (2/10) and infiltrates became smaller Case no 2: A 19-year-old girl, nearsighted with soft contact lenses consulted for a bilateral corneal ulceration and poor vision (1/20). Trophozoit and cysts of Acanthamoeba were found in the contact lens solution. She was treated quickly with Désomédine. Visual acuity improved to 7/10 but the corneal ulceration left a residual opacity     

Fungal keratitis caused by a rare pathogen,Colletotrichum gloeosporioides,in an east coast city of China

BackgroundTo report a case of fungal keratitis caused by Colletotrichum gloeosporioides in an east coast city of China, which are rare pathogens that cause fungal keratitis in humans.MethodsA 52-year-old man whose right eye was injured by a branch of an apple tree during farm work was referred to our Hospital. He was examined by Slit-lamp and the HRT II-RCM confocal scanning microscope, thus suggesting filamentous. Orneal scrapings were acquired and then inoculated into Sabouraud medium incubated at 28 °C and 37 °C. In vitro antifungal susceptibility tests were performed following the CLSI M38-A2 for Filamentous Fungi. Surgical intervention was advised because the abscess in the anterior chamber of the right eye was not completely absorbed.ResultsThe Colletotrichum gloeosporioides isolate was identified by MALDI-TOF mass spectrometry and the BLAST after DNA sequencing of the amplified internal transcribed spacer (ITS) in rRNA. The patient's eye condition is under control and the patient's vision remains at the level of light perception (LP).ConclusionsWe report the rare keratitis caused by C. gloeosporioides in eastern China, which has not been published. Suddenly ocular trauma and old surgical intervention may be the risk factors associated with Colletotrichum keratitis.     

Application of immunoperoxidase staining in the cytodiagnosis of herpes simplex keratitis

In corneal scraping smears from 13 patients with clinically suspected herpes simplex keratitis (HSK), HSK is demonstrated by means of peroxidase-antiperoxidase (PAP) technique with antisera to herpes simplex virus (HSV) in Papanicolaou-destained cellular samples. The staining for HSV antigen was present in seven cases of corneal scraping smears with superficial keratitis (dendritic and geographic ulcers) while six cases of stromal keratitis (deep keratitis) failed to show HSV antigen except in one case. Specific antigen for HSV was predominantly present in the cytoplasm rather than in the nucleus. Immunoreactions were negative with HSV antisera in patients with other infections and in those in a normal control group. Using the PAP technique, detection of HSV antigen in corneal scraping smears was of great value in the diagnosis of HSK, especially in cases of superficial keratitis.     

First case of Tropicoporus tropicalis keratitis in an immunocompetent host from India and review of the literature

Tropicoporus tropicalis is an environmental basidiomycete that has been implicated in nine cases of cutaneous (n = 7) and pulmonary (n = 2) human infections predominantly in chronic granulomatous disease patients. We report here the first case of keratitis caused by Tropicoporus tropicalis in a 40-year-old immunocompetent patient, who presented with sudden diminution of vision in right eye. Corneal scrapings revealed hyaline, septate hyphae in microscopy and culture showed growth of white non-sporulating mycelial growth which was confirmed as Tropicoporus tropicalis by sequencing of ITS region of 28S rDNA. The patient was initiated on topical voriconazole along with natamycin, gatifloxacin and atropine drops. However, despite treatment, corneal ulcer perforated, for which penetrating keratoplasty was performed. Thereafter, he was prescribed amphotericin B (AMB) drops sixteen times a day and ketoconazole 200 mg twice a day with no recurrence reported over one year of follow up. The case represents the first case of infection by this fungus from India and also is the first case to be reported in an immunocompetent host.     

The role of the innate and adaptive immune responses in Acanthamoeba keratitis

Infections of the corneal surface are an important cause of blindness. Protozoal, viral, bacterial, and helminthic infections of the cornea account for up to 9 million cases of corneal blindness. Free-living amoebae of the genus Acanthamoeba produce a progressive infection of the cornea called Acanthamoeba keratitis. Disease is usually transmitted by Acanthamoeba trophozoites bound to soft contact lenses. Infection of the cornea is initiated when the parasite binds to the corneal epithelial surface. Recrudescence can occur and suggests that the adaptive immune response is not aroused by corneal Acanthamoeba infections. Systemic immunization with Acanthamoeba antigens elicits robust Th1 cell-mediated immunity and serum IgG antibody, yet fails to prevent the development of Acanthamoeba keratitis. However, immunization via mucosal surfaces induces anti-Acanthamoeba IgA antibodies in the tears and provides solid protection against the development of Acanthamoeba keratitis. Unlike other immune effector mechanisms that rely on cytolysis, inflammation, release of toxic molecules, or the induction of host cell death, the adaptive immune apparatus prevents Acanthamoeba infections of the cornea by simply preventing the attachment of the parasite to the epithelial surface. The beauty of this mechanism lies in its exquisite simplicity and efficacy.     

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.     

Filter-culture technique using amoeba saline transport medium for the noninvasive diagnosis of Acanthamoeba keratitis

Acanthamoeba, a common free-living amoeba, is increasingly incriminated as a cause of keratitis and corneal ulceration. Between March 1986 and July 1988, specimens from seven patients submitted by ophthalmologists to the City of Milwaukee Health Department's Bureau of Laboratories were culture positive for Acanthamoeba. All patients were contact lens wearers. The specimens were transported at ambient temperature in amoebasaline (5.0 mL) and filtered through 13 mm 0.22 microns cellulose filters. The filters were then plated in cocultivation with Escherichia coli on nonnutrient agar and had positive results for Acanthamoeba in two to five days. Contact lens cases were culture positive for Acanthamoeba in three instances. These results indicate that corneal scraping in amoeba saline transport medium can provide an effective way to diagnose Acanthamoeba keratitis when direct culture of such specimens is not possible.     

Temperature limitation may explain the containment of the trophozoites in the cornea during Acanthamoeba castellanii keratitis

Acanthamoeba keratitis is a serious sight-threatening disease. The relatively low temperature of the cornea may explain why amoebic infections usually are localized in this tissue and rarely spread to other parts of the eye. In this study, the growth rate of the amoeba Acanthamoeba castellanii was examined at different temperatures. The aim was to establish the optimal growth temperature for A. castellanii and to examine the growth within the vicinity of the core body temperature. The growth rates of four clinical and two environmental strains of A. castellanii were estimated at different temperatures, and temperature limitations for the trophozoite stage was established. Movements influenced by temperature gradients were monitored for two clinical strains of A. castellanii. The highest growth rate for each of the six amoebic strains tested was found to be close to 32 °C. The growth of the trophozoites of all examined strains was greatly reduced or completely halted at temperatures above 36 °C and encysted at the elevated temperature. Thus, the optimal growth temperature for the four strains of A. castellanii is close to the surface temperature of the human cornea, while the higher body core-temperature induced encysting of the amoebae. This may explain why most amoebic eye infections are confined to the cornea.     

Analysis of the herpes simplex virus type 1 UL6 gene in patients with stromal keratitis

Recent work suggests that herpes simplex virus (HSV) stromal keratitis in the mouse is caused by autoreactive T lymphocytes triggered by a 16 amino acid region of the HSV UL6 protein (aa299-314), Science 279, 1344-1347). In the present study we sought to determine whether genetic variation of this presumed autoreactive UL6 epitope is responsible for different pathogenic patterns of human HSV keratitis. To accomplish this, we sequenced the HSV UL6 gene from ocular isolates of 10 patients with necrotizing stromal keratitis, 7 patients with recurrent epithelial keratitis, and 8 patients with other forms of HSV keratitis. The sequences obtained predicted identical UL6(299-314) epitopes for all 25 viral isolates. Furthermore, the upstream sequence of all isolates was free of insertions, deletions, and stop codons. We conclude that different pathogenic patterns of human HSV keratitis occur independent of genetic variation of the HSV UL6 (299-314) epitope.     

Impaired innate immunity of ocular surface is the key bridge between extended contact lens wearing and occurrence of Acanthamoeba keratitis

Acanthamoeba keratitis is a progressive, sight-threatening corneal disease. Extended wearing contact lens is one of predisposed factors. Early studies mostly focused on "improper contact-lens hygiene", which described that contact lens wearers have more opportunities to contact with pathogens directly and prone to get A. keratitis. However, improper contact-lens hygiene can not explain the phenomenon that Acanthamoeba protozoon were found in normal individuals' lens-cases. So there might be other factors related with A. keratitis. Recently, more attention has been paid on the influence of extended wearing contact lens on the innate immunity of ocular surface. It has been proven that in contact lens wearers the reactivity of polymorphonuclear leucocytes (PMNs) and the concentration of certain inflammatory mediators were significantly altered compared with that in non-lens wearers. Moreover, other studies showed the important contributions of innate immunity on occurrence and development of A. keratitis. With the contribution of extended wearing contact lens on immunity and the relation between innate immunity and Acanthamoeba, we suggest that the impaired innate immunity of ocular surface may be a key bridge between extended wearing contact lens and A. keratitis. With the impaired innate immunity caused by extended contact-lens wearing, the Acanthamoeba trophozoites and cysts could not be easily killed, therefore A. keratitis was occurred and aggravated. Understanding the immunological mechanism of extended contact lens wearing on the A. keratitis may give more contributions on the research of the disease, and facilitate the production of contact lens with much higher biocompatibility.     

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.