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Pathogenesis of microbial keratitis
Accepted Manuscript Pathogenesis of microbial keratitis Sahreena Lakhundi, Ruqaiyyah Siddiqui, Naveed Ahmed Khan PII:
S0882-4010(16)30758-6
DOI:
10.1016/j.micpath.2016.12.013
Reference:
YMPAT 2034
To appear in:
Microbial Pathogenesis
Received Date: 8 November 2016 Revised Date:
30 November 2016
Accepted Date: 5 December 2016
Please cite this article as: Lakhundi S, Siddiqui R, Khan NA, Pathogenesis of microbial keratitis, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2016.12.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pathogenesis of microbial keratitis
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Sahreena Lakhundi, 2Ruqaiyyah Siddiqui, 2Naveed Ahmed Khan*
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Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan;
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Department of Biological Sciences, Faculty of Science and Technology, Sunway University,
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Selangor, Malaysia.
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Short title: Microbial keratitis
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*Corresponding address: Department of Biological Sciences, Faculty of Science and
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Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext: 7176.
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Fax: 60-(0)3-5635-8630. E-mail: [email protected]
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Abstract Microbial keratitis is a sight-threatening ocular infection caused by bacteria, fungi, and protist pathogens. Epithelial defects and injuries are key predisposing factors making the eye
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susceptible to corneal pathogens. Among bacterial pathogens, the most common agents
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responsible for keratitis include Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus
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pneumonia and Serratia species. Fungal agents of corneal infections include both filamentous as
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well as yeast, including Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia, Paecilomyces,
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Scedosporium and Candida species, while in protists, Acanthamoeba spp. are responsible for
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causing ocular disease. Clinical features include redness, pain, tearing, blur vision and
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inflammation but symptoms vary depending on the causative agent. The underlying molecular
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mechanisms associated with microbial pathogenesis include virulence factors as well as the host
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factors that aid in the progression of keratitis, resulting in damage to the ocular tissue. The
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treatment therefore should focus not only on the elimination of the culprit but also on the
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neutralization of virulence factors to minimize the damage, in addition to repairing the damaged
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tissue. A complete understanding of the pathogenesis of microbial keratitis will lead to the
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rational development of therapeutic interventions. This is a timely review of our current
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understanding of the advances made in this field in a comprehensible manner. Coupled with the
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recently available genome sequence information and high throughput genomics technology, and
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the availability of innovative approaches, this will stimulate interest in this field.
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Key words: Cornea; Keratitis; Protist; Bacteria; Yeast
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1. Introduction The unique structure of the human eye along with its exposure to environment, renders it susceptible to a number of agents responsible for causing infection. Injuries and epithelial defects
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impair defense mechanisms and exposure to pathogenic microbes can lead to corneal
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inflammation or keratitis. The intact ocular surface thwarts most microorganisms but once
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anatomical barriers are breached, host defenses against pathogens are less than sufficient to
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prevent infection that can lead to eventual loss of vision. Microbial or infectious keratitis is a
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potentially sight-threatening ocular condition caused by bacteria, fungi, protists etc. It is the
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inflammation of the cornea caused by pathogenic microbes that eventually invades the corneal
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stroma causing inflammation, and ultimately destruction of these structures (Ansari et al., 2013;
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Wong et al., 2012). The most common pre-disposing factors to develop infectious keratitis
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include the use of contact lenses, especially overnight or extended wear lenses, inadequate
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disinfecting solutions, trauma, ocular surgery especially corneal surgery, chronic ocular surface
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disease, systemic disease like diabetes mellitus and/or extended use of topical corticosteroids
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(Stapleton et al., 2012; Wong et al., 2012). Patients usually present with redness, tearing, rapid
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onset of pain and blur vision. Clinical presentation may vary depending on the causative agent
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responsible for causing keratitis. The condition should be treated as a medical emergency and
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adequate treatment should commence promptly. If appropriate antimicrobial treatment is
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delayed, only 50% of the eye gains good visual acuity (Jones, 1981). Appropriate management
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and timely onset of treatment can reduce the incidence of severe visual loss restricting corneal
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damage. Here, we present a concise review of the bacterial, fungal and protist keratitis.
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Numerous microorganisms can infect the eye either by direct or indirect introduction into the
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eye. The most common clinically important microorganisms involved in eye infections are
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reviewed in this article with relation to the anatomical part of the eye involved in the disease,
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along with a discussion of the pathogenic mechanisms and management of the disease.
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2. Acanthamoeba keratitis
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Acanthamoeba keratitis is a rare but sight threatening corneal infection, caused by an
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opportunistic protist pathogen belonging to the genus Acanthamoeba. They are ubiquitous, free-
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living protists dispersed in a variety of environments including air, soil, freshwater, tap water,
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hospital equipments, surgical instruments, showers, ventilation ducts, air-conditioning units,
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chlorinated swimming pools, sewage etc. (De Jonckheere, 1991; Kilvington & White, 1994).
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Phenotypic switching into a cyst form enables Acanthamoeba to withstand adverse
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environmental conditions. The Acanthamoeba trophozoite has an amoeboid shape that contains
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spike-like structures known as acanthopodia and during this stage, amoebae feed and reproduce
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under favourable environmental conditions (Marciano-Cabral & Cabral, 2003; Visvesvara et al.,
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2007). However, under extreme situations such as lack of nutrients, hyperosmolarity,
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desiccation, extreme pH, temperatures, and the presence of antimicrobials; the trophozoite
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rounds up and confines itself within a double-walled resistant cyst form that has minimal
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metabolic activity. The cyst stage presents a major problem in the successful treatment of
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Acanthamoeba infections as they are resistant to various antimicrobial agents, often leading to
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recurrence of the disease upon discontinuation of therapy (Niederkorn et al., 1999). Although
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exposure to Acanthamoeba spp. appears to be common due to its ubiquitous nature, the incidence
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of Acanthamoeba keratitis is less common. The main risk factors for Acanthamoeba keratitis are
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contact lens wear for extended periods, corneal trauma, non-sterile contact lens rinsing,
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swimming while wearing contact lenses and biofilm formation on contact lens (Marciano-Cabral
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& Cabral, 2003; Trabelsi et al., 2012). While contact lens wear is the leading risk factor,
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Acanthamoeba can cause keratitis in non-contact lens wearers as well (Dart et al., 2009).
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However, it is often overlooked in non-CLs users as a causative agent of keratitis, where it is
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usually associated with trauma and/or exposure to contaminated water, soil and organic matter.
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In addition, Acanthamoeba keratitis has been reported after invasive or radial keratoplasty and/or
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laser-assisted in situ keratomileusis (LASIK) where the lesions generally worsen due to a delay
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in the diagnosis and treatment (Garg et al., 2010; Sharma et al., 2011).
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Clinical manifestations of Acanthamoeba keratitis include excruciating pain characterized
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by redness, epiphora, lacrimation, eyelid diptosis, conjonctival hyperhemia, foreign body sensation and photophobia (Niederkorn et al., 1999; Mahgoub, 2010; Alizadeh et al., 1994). As
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the disease progresses, stromal involvement results in infiltration of inflammatory cells
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displaying a characteristic ring infiltrate. If not promptly diagnosed and treated aggressively,
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cornea becomes ulcerated leading to perforation, ring infiltrate, stromal abscess formation, loss
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of visual acuity and eventually blindness and enucleation. Acanthamoeba was first recognized as an ocular pathogen in 1973 in the U.S.A where the
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first case was reported by an ocular microbiology group in Dallas (Clarke et al., 2012). However,
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the first published report of Acanthamoeba keratitis emerged in the UK in 1974 and was
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associated with minor eye injury (Naginton et al., 1974). The first case in a contact lens wearer
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was reported 10 years later in 1984 from a patient wearing soft contact lenses (Samples et al.,
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1984). Since then the number of cases has been on the rise especially in recent years, mainly due
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to an increase in the number of contact lens users, better diagnostics and increased awareness.
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2.1.
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2.1.1. Adhesion The first step in the pathogenesis of Acanthamoeba keratitis is the ability of amoebae to
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Pathogenesis
bind to the corneal epithelium, a factor that determines the degree of pathogenicity of different
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isolates. The pathogenic cascade begins via amoebal binding to mannose glycoproteins on the
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corneal surface through adhesin expressed on the trophozoite membrane called the mannose-
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binding protein (MBP) (Panjwani, 2010; Clarke & Niederkorn, 2006; Garate et al., 2004). This
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adherence is a crucial prerequisite for producing infection. In addition. mild trauma or corneal
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abrasion is required for the establishment of corneal infection. Abrasion or trauma results in
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increased expression of mannose glycoproteins on the corneal epithelium and hence there is
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increased adhesion of amoebae to the damaged cornea, compared with the healthy cornea
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(Clarke & Niederkorn, 2006). The contact lenses, in addition to serving as a vector for the
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introduction of trophozoite onto the corneal surface, also up regulate mannose glycoproteins on
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the corneal epithelium. This results in an increased number of trophozoites binding to the contact
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lens-conditioned cornea compared to the normal cornea thus leading to findings that more than
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80% of Acanthamoeba keratitis cases are associated with the use of CLs (Alizadeh et al., 2005).
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The second important element involved in adhesion is the number of acanthopodia on the
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amoebic surface. Pathogenic amoebae have more than 100 acanthopodia/cell compared to non-
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pathogenic amoebae. Therefore, non-pathogenic amoeba present very low binding levels to host
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cells compared to pathogenic amoebae (Khan, 2001; Panjwani, 2010). Hence the number of
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acanthopodia is also believed to be closely related to the rate of adhesion to the corneal surface.
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Once attached, intracellular signaling processes trigger the pathogenic cascade involved in the
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pathogenesis of Acanthamoeba keratitis.
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2.1.2. Cytopathic effect
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Trophozoite binding to corneal surface is tailed by extensive desquamation of the corneal epithelium, leading to penetration of the underlying Bowman’s membrane. Trophozoite-
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mediated cytopathic effect proceed via several mechanisms such as direct cytolysis, phagocytosis
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and apoptosis (Alizadeh et al., 1999). It has been observed that Acanthamoeba-mediated direct
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cytolysis is dependent on calcium channel activity and on cytoskeletal elements, as the inhibition
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of both via the calcium channel blocker, Bepridil and actin polymerization inhibitor, cytochalasin
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D overcomes Acanthamoeba-mediated cytolysis by over 98% (Taylor et al., 1995). In addition
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the presence of structures like food cups or amoebastomes on the surface of Acanthamoeba
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suggests that the amoeba binding to host cells leads to secondary events like phagocytosis by
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which the amoeba bites or engulfs host cells. It has been observed that Acanthamoeba
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phagocytose and/or engulfs corneal epithelial cells and that this activity is mediated via
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amoebastomes present on the surface of amoebae (Khan, 2001).
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Apart from inducing direct cell death, several studies indicate that Acanthamoeba induces apoptosis of keratocytes, iris ciliary body cells, retinal pigment epithelial cells, corneal epithelial
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cells, neuroblastoma cells etc. (Marciano-Cabral & Cabral, 2003; Niederkorn et al., 1999; Clarke
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et al., 2005; Stopak et al., 1991). Acanthamoeba induces membrane blebbing, formation of
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apoptotic bodies, DNA laddering, nuclear chromatin condensation of host cell, all known
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markers for apoptosis (Alizadeh et al., 1994; 1994a; Pettit et al., 1996). It has been observed that
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exposure to mannose stimulates the production of a 133-kDa protease termed mannose-induced
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protein (MIP133) by Acanthamoeba trophozoites. This production of MIP133 appears to be
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another vital element of the pathogenic cascade of Acanthamoeba keratitis. It initiates apoptosis
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of corneal epithelial cells in a caspase-3-dependent pathway in vitro and it is possible that the
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trophozoite uses this pathway in the desquamation of the corneal epithelial cells in vivo (Hurt et
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al., 2003a; Hurt et al., 2003a). Furthermore, clinical isolates of Acanthamoeba but not soil
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isolates produce MIP133 and can cause disease in animals, signifying an association between
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MIP133 production and pathogenic potential. Hence the trophozoite-mediated cytopathic effect
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to destroy epithelial cells occurs by three independent mechanisms i.e., direct cytolysis,
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phagocytosis and apoptosis.
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2.1.3. Stromal invasion
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Following binding and desquamation of the corneal epithelium, the next step in Acanthamoeba keratitis involves the penetration and dissolution of the underlying collagenous
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stroma. The Acanthamoeba trophozoites secrete a variety of proteases with nonspecific
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collagenolytic activity to facilitate the invasion and degradation of stroma. These include serine
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proteases, cysteine proteases, metalloproteinases, elastase, collagenolytic enzyme, phospholipase
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A and a novel plasminogen activator (Cao et al., 1998; Cho et al., 2000; Hadas & Mazur, 1993).
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Studies have shown the direct role of extracellular proteases in Acanthamoeba keratitis by co-
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incubating corneal epithelial cells with Acanthamoeba conditioned medium, which resulted in
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host cell cytotoxicity (He et al., 1990). This cytotoxic effect was abolished in the presence of a
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serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), suggesting a crucial role played
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by serine proteases in the pathogenicity of Acanthamoeba keratitis (Dudley et al., 2008).
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Furthermore, pathogenic Acanthamoeba exhibit higher protease activity compared to non-
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pathogenic amoebae (Khan et al., 2000). Moreover, serine proteases are also able to degrade
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collagen, fibronectin, laminin, secretory IgA, IgG, plasminogen, BSA, fibrin, fibrinogen, and
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rabbit corneal proteins (Siddiqui & Khan, 2012). These properties of proteases are important
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because collagen type I is the main structural protein in the corneal stroma which is important for
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the maintenance of its integrity. In addition sIgA, the main immunoglobulin in tears and a
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primary barrier against pathogenic microbes are also degraded by these proteases. Other
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extracellular enzymes like elastase and phospholipase A are capable of degrading connective
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tissues. These findings suggest that collagenolytic enzymes have a role in corneal lesions and the
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generation of ring-like stromal infiltrates which are characteristic features of Acanthamoeba
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keratitis (He et al., 1990).
Pathogenic Acanthamoeba can also use plasminogen activator that is constitutively present in ocular isolates, to catalyze the cleavage of host plasminogen to plasmin, which in turn
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activates matrix metalloproteinases (MMPs) to degrade components of extracellular matrix
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(Mitra et al., 1995; Berman, 1993). They result in ulceration of normally quiescent cornea by
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degrading the components of basement membrane and extracellular matrix, including type I and
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II collagens, laminin and fibronectin (Parks, 1999). Corneal stroma, unlike other extracellular
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matrices is vital for the normal functioning of the eye. Its degradation affects normal vision and
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can lead to blindness. Studies have also shown that pathogenic isolates of Acanthamoeba are
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able to secrete ecto-ATPases which play a role in protecting the cells from effector cells of the
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immune system. Secondly, they play a role in repolarization of the membrane after
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depolarization by an active compound by acting as a proton pump. Lastly, these enzymes are
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thought to be related to adherence to the host cell (Siddiqui & Khan 2012).
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2.1.4. Neuritis Acanthamoeba spp. are highly motile and secrete a plethora of proteases that augment their
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pathogenesis by desquamating corneal epithelium, penetrating Bowman’s membrane and
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invading the corneal stroma. This cascade of events comes to a halt and trophozoites almost are
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never able to penetrate the corneal endothelium and the anterior chamber of the eye (Clarke &
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Niederkorn, 2006). However, they seem to cluster around the corneal nerves producing radial
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keratoneuritis (Alizadeh et al., 2005). Studies have demonstrated the chemotactic response of
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Acanthamoeba trophozoites towards extracts of neuronal cells and neural-crest-derived cells but
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not towards the corneal epithelial or stromal cells (Pidherney et al., 1993). Moreover, in vitro
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studies have also demonstrated the killing of nerve cells by trophozoites via direct cytolysis
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and/or apoptosis (Pettit et al., 1996). These cytopathic effects contribute to nerve damage and
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may account for the excruciating pain experienced by the patient, coupled with this infection in
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vivo.
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2.2.
Currently the treatment for Acanthamoeba keratitis involves topical application of cocktail
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Treatment
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of drugs, as there is no single effective drug treatment against Acanthamoeba keratitis. This is
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mainly because of the degree of virulence of the infecting isolate which is different for each
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individual case and therefore makes it impossible to establish a correlation between in vitro and
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in vivo efficacies. The best therapeutic outcomes are expected only when, it is diagnosed early,
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treated adequately and aggressively with a high level of patient compliance. Acanthamoeba
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trophozoites are sensitive to most available drugs; however the cysts present a major issue in the
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course of treatment and are the main reason for the recurrence of infection upon discontinuation
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of therapy. Thus, successful management most often necessitates extended therapy with a
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combination of well tolerated drugs with minimal toxicity to hosts cells that results in a
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favourable visual consequences with reduced need for surgical interventions.
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The most effective topical agents currently used against Acanthamoeba trophozoites and cysts are the biguanides (polyhexamethylene biguanide (PHMB) 0.02 – 0.06% or chlorhexidine
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0.02 – 0.2%) in combination with diamidine (propamidine isethionate 0.1% or hexamidine 0.1%)
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(Wright et al., 1985; Khan, 2003; Dart et al., 2009; Lorenzo-Morales et al., 2013). The former
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drugs are membrane-acting cationic biocides that interact with negatively charged surface
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proteins of Acanthamoeba resulting in leakage of the cellular components (Perrine et al., 1995).
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The latter however exert their amoebicidal effects via cationic surface-active properties inducing
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structural changes resulting in cell permeability and are effective DNA synthesis inhibitors
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(Duguid et al., 1997). In vitro studies revealed that cationic agents have the best and most
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constant amoebicidal and cysticidal activity (Elder et al., 1994; Hay et al., 1994). If these drugs
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are applied early on in the development of infection, at high frequency along with neomycin and
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0.15% dibromopropamidine, they show successful prognosis (Lorenzo-Morales et al., 2013).
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Hourly drops of PHMB and hexamidine day and night may be tapered off after 48 hours to
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alleviate epithelial toxicity, to hourly drops during the day for the next 72 hours. This therapy
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reduces viable trophozoites when they are more susceptible and prevent them from turning into
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fully mature cysts. This treatment is then reduced to 2-hourly application during the day for the
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next 3 – 4 weeks and then gradually tailored depending on the individual case. Treatment
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regimes on average last over a period of 6 months, ranging from 0.5 to 29 months (Radford et
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al., 1998).
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In the case of inflammation along with persistent infection, corticosteroids have been implicated however, there use is controversial as they cause suppression of host immune
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response (Lorenzo-Morales et al., 2013). Recent reports also point towards the use of the azoles
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as an adjuvant to biguanide and diamidine therapy in resistant Acanthamoeba keratitis cases
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(Bang, et al., 2010). Therefore topical and intrastromal voriconalzole drops (1%) have been used
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successfully in 3 cases with resistant Acanthamoeba keratitis. Surgical management via epithelial
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debridement, corneal graft surgery (keratoplasty) and/or DALK (deep lamellar keratoplasty) may
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be required in some cases where the corneas are permanently scarred or where the topical and
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oral treatments have failed (Illingworth & Cook, 1998; Chen et al., 2004; Sony et al., 2002;
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Jiang et al., 2006; Parthasanrathy & Tan, 2007). Recently, photorefractive surgery seems to be a
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very promising modality and have been used in four cases of early stage Acanthamoeba keratitis,
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where the patients developed large corneal abscesses in the upper third thickness of stroma
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(Kandori et al., 2010). Another relatively new approach is collagen cross-linking which has
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shown promising results in clinically (Garduno-Vieyra et al., 2011; Khan et al., 2011; Muller et
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al., 2012). It is presumed that collagen probably prevents further tissue damage and prevents
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reproduction of amoebae and hence the patients showed rapid reduction in ocular symptoms and
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ulcer size.
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Moon et al., (2015) recently reported that autophagy inhibitors have anti-amoebic effects
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and if used with low concentrations of PHMB (0.00125%) has very low cytopathic effect on
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human corneal cells and a high cytopathic effect on Acanthamoeba cells. In addition, Aqeel et
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al., (2015) suggested photochemotherapeutic strategy against Acanthamoeba infections and
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showed that mannose-conjugated porphyrin has the potential for targeted photodynamic therapy
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of Acanthamoeba infections. Regardless of significant advances in Acanthamoeba keratitis 12
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treatment over the last decade or so, prevention still seems to be the best option. In addition,
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contact lens wearers must be thoroughly educated on the proper hygiene to handle contact lenses
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to avoid this serious sight-threatening infection.
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3. Mycotic keratitis
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Mycotic keratitis is the fungal infection of the cornea caused by either filamentous and/or
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yeast-like fungi. They may account for more than 50% of all culture-positive microbial keratitis
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especially in tropical and sub-tropical countries (Xie et al., 2006; Nath et al., 2011; Thomas &
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Kaliamurthy, 2013). A strong geographical correlation has been reported to exist between the
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occurrences of different types of keratomycosis. For example, the proportion of keratitis due to
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yeast-like fungi show a tendency to increase towards temperate climates whereas corneal ulcers
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caused by filamentous fungi appear to be more common towards tropical latitudes (Leck et al.,
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2002).
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Filamentous fungi such as Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia, Paecilomyces and Scedosporium apiospermum are most commonly associated with keratitis
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caused by filamentous fungi (Thomas & Kaliamurthy, 2013). However Candida albicans and
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other Candida species are most common keratitis-causing yeast-like fungi. Former is reported to
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be due to ocular trauma which is usually the most important predisposing factor in healthy young
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males engaged in agricultural or other outdoor activities (Nath et al., 2011; Gopinathan et al.,
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2009; Thomas & Kaliamurthy, 2013). These fungi do not invade the intact cornea and
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penetration only occurs once the epithelium is abraded. Traumatizing agents of animal origin or
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vegetative matter, soil or dust particle either directly implant fungal conidia on abraded corneal
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epithelium for fungal invasion (Nath et al., 2011; Gopinathan et al., 2009; Thomas &
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Kaliamurthy, 2013). The use of corticosteroids, ocular surgery, ocular surface disease and
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contact lens wear has now also been increasingly identified as a significant risk factor.
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Conversely, C. albicans and related fungi are only linked to keratitis when there is a pre-existing
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ocular condition like insufficient tear secretion, defective eye closure, or some systemic illness
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such as diabetes mellitus or immunosuppression (Thomas, 2003). This form of keratomycosis
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may also supervene on a pre-existing epithelial defect caused by herpes keratitis or abrasion due
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to contaminated contact lenses.
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Fungal infections of the cornea need to be promptly addressed to facilitate full recovery. In
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case of filamentous fungal keratitis, clinical manifestations include sudden onset of pain along with photophobia, discharge with reduced vision and opacity on the surface of the cornea
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suggestive of an ulcer (Ansari et al., 2013). It may involve any part of the cornea and show firm,
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sometimes dry elevated slough, hyphate lines extending into the normal cornea beyond the edge
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of the ulcers, multifocal granular or feathery grey-white satellite stromal infiltrates, immune ring,
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Descemet’s fold and mild iritis (Thomas, 1994; Rosa et al., 1994; Srinivasan, 2004; Thomas &
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Kaliamurthy, 2013). Although each fungal keratitis exhibits these basic features, they may vary
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depending on the etiological agent. Severe, chronic filamentous keratitis somewhat resemble
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bacterial suppuration and may involve the entire cornea. However those due to yeast-like and
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related fungi may resemble bacterial keratitis with an overlying epithelial defect, discrete
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infiltrate and slow progression (Sun et al., 2007).
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3.1 Mechanism
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3.1.1
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Adhesion
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Interaction of pathogenic fungi with host cells is the key factor in the pathogenesis of mycotic keratitis. Adherence of microorganisms to host cells is a pre-requisite for the initiation
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of the infection. Hence fungal infections of the cornea begin via adhesion of fungi to the
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damaged cornea. Fungal pathogens display a variety of adhesins that are capable of adhering to
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various cell types and interact with a variety of host proteins and glycoproteins present in host
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cells (Hostetter, 1994). The outer fibrillar layer of yeast and filamentous fungal cell wall is
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composed of mannan or mannoprotein (Imwidthaya, 1995; Thomas, 2003). The adhesive
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mannoproteins play an essential role in fungal binding to corneal tissues. These lectin-like
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proteins recognize D-mannose or mannose glycoproteins on the surface of corneal epithelial
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cells. Damage to the cornea results in the upregulation of mannose glycoproteins on the corneal
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surface (Clarke & Niederkorn, 2006); it may therefore play an important role in the adhesion and
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pathogenesis of fungal keratitis. Ocular trauma is the most important pre-disposing factor in
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mycotic keratitis (Nath et al., 2011; Gopinathan et al., 2009; Thomas & Kaliamurthy, 2013); the
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absence of which prevents fungal penetration into the cornea. Thus, the correlation of ocular
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trauma with an increased expression of mannose glycoproteins on the corneal surface and
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presence of adhesive mannoproteins on the surface of fungal cell wall is likely involved in the
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pathophysiology of fungal keratitis.
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The corneal epithelium also possess other potential fungal binding sites like laminin,
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fibronectin, collagen etc. which also play a role in keratitis (Thomas, 2003). Tronchin et al.,
20
(1988) demonstrated the importance of an outer fibrillar layer of the germ tube in the process of
21
adhesion to plastics. Bouchara et al., (1990) reported that these major components of the fibrillar
22
cell wall layer of the germ tube act as a receptor for laminin, fibrinogen and mediates its
23
attachment to membranes. The involvement of specific interaction of Aspergillus fumigatus with 15
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finrinogen and its role in cell adhesion has shown that fungal conidia adhere avidly to wells
2
coated with laminin, fibrinogen, collagen and fibronectin substrates (Coulot et al., 1994). This
3
was further supported by Bromley & Donaldson (1996), who showed the ability of fibrinogen
4
and laminin to inhibit the adherence of conidia to pulmonary epithelial cell lines. In addition
5
sialic-acid dependent lectins have also been reported to play an important role in recognition of
6
laminin and fibrinogen by Aspergillus and Penicillium conidia.
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Furthermore, the outer layer of conidia also plays a crucial role in the early stages of
8
infectious process. The surface of resting conidia has been reported to have proteins belonging to
9
the hydrophobin family (Parta et al., 1994; Thau et al., 1994) which are detected in all
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filamentous fungi and thought to play a role in adherence (Scholtmeijer et al., 2001).
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3.1.2
The etiological agent in Fusarium keratitis is able to invade the cornea gaining access to
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Invasiveness and Morphogenesis
the anterior chamber of the eye. There, at the pupillary area, it forms a lens-iris-fungal mass
14
affecting the normal drainage of aqueous humor leading to an increase in the intraocular pressure
15
causing fungal malignant glaucoma (Kuriakose & Thomas, 1991). Initially, malignant glaucoma
16
was thought to occur only in Fusarium keratitis, however recently Jain et al., (2007), reported a
17
case of Aspergillus-induced malignant glaucoma where the uniform shallowing of the anterior
18
chamber was present with raised IOP unresponsive to antiglaucoma measures. Eventually A.
19
flavus was isolated from the anterior chamber of the eye.
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Fungal invasiveness is directly related to the fungal load and inversely proportional to the
21
intensity of inflammatory response (Vemuganti et al., 2002). Therefore, the heavier the load of
16
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the fungus, the greater the extent of invasion and the smaller the number of inflammatory cells.
2
In early mycotic keratitis, the heavy fungal load with deep tissue penetration may overcome the
3
inflammatory response in corneal tissue, encouraging the progression of the disease. Conversely
4
the inflammatory response in early keratitis may be mild and therefore the fungi multiply
5
extensively and penetrate deep into the tissues. The putative sequence of events in which it
6
occurs still needs to be confirmed.
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Phenotypic switching or morphogenesis is an important method of adaptation used by
8
some microbes in different microenvironments to survive inside infected hosts. This permits
9
fungi to survive in the presence of anti-fungal drugs and resist anti-microbial therapy. The same
10
intrahyphal hyphae or hypha-in-hypha and thickened fungal cell walls are seen in corneal tissues
11
infected with L. theobromae and in corneas infected with F. solani keratitis treated earlier with
12
corticosteroids, as seen in the presence of antifungal drugs (Thomas et al., 1991; Kiryu et al.,
13
1991). This suggests that these morphological alterations may allow fungi to evade host defenses
14
and present a barrier against antifungal drugs. Jackson et al., (2007) demonstrated the role of
15
EFG1-regulated SAP6 gene of C. albicans which encodes for a unique secreted aspartyl
16
proteinase. They concluded that proteases contribute to corneal pathogenicity in C. albicans
17
keratitis and are also associated with morphogenic transformation from yeast to invasive
18
filamentous forms.
19
3.1.3
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Toxigenicity
Various keratitis causing fungi are known to produce mycotoxins; however their exact role
21
in the pathogenesis of keratitis is still unknown. Several toxins produced by Fusarium spp.
22
include nivalenol, T-2 toxin, deoxynivalenol, diacetoxyscirpenol and fusaric acid. Raza et al., 17
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(1994) studied the relationship of toxin production with the severity of disease and concluded
2
that toxin production in vitro did not relate to the clinical presentation of severity of keratitis and
3
the outcome of the treatment. The toxic effects of aflatoxins on the cornea of chicks has been
4
established (Kant et al., 1984). It was demonstrated that when aflatoxin was administered to
5
chicks, haziness of the cornea and separation of corneal lamellae was observed in addition to
6
infiltration by polymorphonuclear leukocytes. Leema et al., (2010) demonstrated that A. flavus
7
isolated from keratitis patients produce significantly more aflatoxin compared to those of the
8
environmental isolates. However the reason behind this phenomenon is unclear and requires
9
further study.
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The ability of fungi to produce various enzymes could also damage tissues, facilitate invasion and eventually influence the severity and outcome of the disease. Zhu et al., (1990)
12
examined the role of fungal proteases in the pathogenesis of mycotic keratitis. It was observed
13
that the clinical isolate of A. flavus, isolated from a patient with severe keratitis, secrete variety
14
of proteases including serine proteases, cysteine proteases, metalloproteases and concluded that
15
fungal collagenases are the mediator of the severe corneal destruction observed in keratitis.
16
When attempted to compare the presence of fungal proteases in vitro and in vivo, the corneal
17
isolates of A. flavus and F. solani predominantly secreted serine proteases and little
18
metalloproteases in vitro. In vivo, however the protease profile shifted to metalloproteases and no
19
serine protease activity was detected (Gopinathan et al., 2001). Matrix metalloproteinases are
20
thought to play a pathological role in the degradation of extracellular matrix components such as
21
basement membrane collagen; same as those found in corneal basement membrane and stroma
22
(Boveland et al., 2010). They have also been shown to be upregulated in ulcerative fungal
23
keratitis in rabbit as well as in the tear film of horses with ulcerative keratitis and hence are
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thought to play a probable role in the pathogenesis of fungal keratitis (Gopinathan et al., 2001;
2
Ollivier et al., 2004). However, the exact role of fungal proteases in mycotic keratitis needs
3
further investigations.
4
3.2 Treatment
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Fungal keratitis is a complex entity with many considerations when it comes to treatment.
6
On the whole, treatment generally entails of chemotherapy consisting of topical and/or systemic
7
drugs, alone or in combination with surgical treatment. However, in developing countries with
8
limited care and economical barriers, mycotic keratitis is of major concern as it can cause visual
9
loss in a demographic population that has limited access to care. Each antifungal has its own
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benefits and limitations and therefore must be selected carefully based on etiological agent and
11
susceptibility testing. There are several classes of antifungals available like polyenes, azoles and
12
fluorinated pyrimidines, each with their own advantages and disadvantages. Polyenes, including
13
natamycin, nystatin and amphotericin B, disrupt fungal cell by binding to the cell wall ergosterol
14
of both filamentous as well as yeast-like fungi. However their tissue penetrating ability is poor
15
and therefore is mostly recommended in cases of superficial corneal infections (Thomas, 2003;
16
Ansari et al., 2013). Administration is every 30 min during the first 24 hours and then every hour
17
for the next 24 hours and then gradually tapered off according to the response. While
18
amphotericin B is commonly administered as a topical solution, intracameral administration is an
19
effective alternative in reducing time to disappearance of hypopyon and final improvement
20
(Yoon et al., 2007). Although active against both forms of fungi, it has shown variable activity
21
against Fusarium species. Owing to its side effect profile and lack of coverage against Fusarium
22
keratitis, it is not considered as a first line agent (Ansari et al., 2013). Natamycin on the other
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hand has a broad-spectrum activity against filamentous fungi and is the first line treatment
2
against fungal keratitis and the drug of choice for Fusarium keratitis. The drug can only be given
3
topically and hence can only treat superficial fungal keratitis as opposed to deep stromal fungal
4
invasion. The presence of deep lesions may therefore require other antifungals like azoles
5
administered via subconjunctival or intravenous routes (Tanure et al., 2000).
The azoles including imidazoles and triazoles, at low concentrations inhibit ergosterol
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biosynthesis while, cause direct damage to cell walls at higher concentrations (Tatsumi et al.,
8
2013). Fluconazole and ketoconazole show good intraocular penetration and are therefore good
9
agents against keratitis with deep lesions (Panda et al., 1996). They are the preferred treatment
10
against both candida and filamentous fungi however, fluconazole has narrow coverage against
11
filamentous organisms but ketoconazole is active against Aspergillus, Candida as well as
12
Curvularia species (Rao et al., 1997; Fitzsimons & Peters, 1986; Ansari et al., 2013).
13
Voriconazole is a good alternative with minimal toxicity and is not only active against Candida
14
but also against filamentous fungi such as Fusarium spp. (Marangon et al., 2004; Lalitha et al.,
15
2007). In refractory FK cases, topical voriconalzole has been used as an adjunct to natamycin
16
along with intrastromal injections of voriconalzole with success (Hariprasad et al., 2008).
17
Hariprasad et al., (2008) reviewed 40 case-reports and concluded that voriconalzole is a safe
18
alternative against major ocular fungal infections but shouldn’t be used as a single agent for
19
initial treatment.
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The fluorinated pyrimidines such as flucytosine etc. are converted into thymidine analog
21
blocking fungal thymidine synthesis. They are usually administered with an azole or
22
amphotericin B for synergistic effects and to avoid development of resistance. Subconjunctival 20
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injections have also been used in patients with severe keratitis. Regardless of the drug choice,
2
successful therapy requires prolonged frequent drug administration and therefore adherence of
3
patients to treatment is also an important factor affecting the outcome of treatment. In addition,
4
liposomal preparation of anti-fungal drugs are also under trail and has demonstrated some
5
efficacy in rabbit models (Habib et al., 2010; Abdel-Rhaman et al., 2012). Furthermore Rasool et
6
al., (2012) compared the efficacy of topical clotrimazole-β-cyclodextrin (CBC), a comparatively
7
new anti-fungal compared to amphotericin B and concluded that CBC reduces the period of
8
treatment on average by a duration of 1 week. The authors also reported greater efficacy of CBC
9
in cases of Candida keratitis.
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Surgical interventions are required in case of complications of acute infectious processes as well as if the disease is refractory to medical management. Penetrating keratoplasty (PK) is the
12
most common surgical treatment used to excise lesions of the cornea and replaced with a donor
13
corneal graft in case of refractory or severe cases of fungal keratitis (Prakash et al., 2008). In
14
addition, it is an effective way to treat corneal perforations as a result of fungal infections (Xie et
15
al., 2007). As an alternative to PK, debridment is also used where causative agent and
16
necrotizing material is removed thereby enhancing the penetration of anti-fungal medications by
17
the removal of epithelium. It is recommended where the necrotizing tissues are hindrance to the
18
healing of corneal ulcers.
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Alternate surgical procedure includes lamellar keratoplasty (LK) in which only diseased
20
layers of the corneal surface are excised thereby leaving the underlying structures of the cornea
21
intact. It is recommended when the fungal invasion is only focal (Price et al., 2005; Reddy et al.,
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1
2013). In thisway, the risk of endothelial rejection, the most severe type of corneal graft
2
rejection, is minimized. The challenging nature of fungal keratitis along with less than ideal outcome of current
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treatment regimes, various experimental advances have also been made. Corneal collagen cross-
5
linking (CXL) is a relatively new technique currently been investigated in conjunction with
6
photo-activated riboflavin (PAR) in refractory keratitis cases (Li et al., 2013; Panda et al., 2012).
7
Investigations into the use of nanoparticle technology with terbinafine and silver have also
8
shown promising results (Tayel et al., 2013; Xu et al., 2013). In addition, topical aqueous garlic
9
extracts have been tested on rabbit keratitis models infected with A. flavus (Ismaiel et al., 2012).
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Nonetheless, fungal keratitis is a complex entity with many considerations when it comes to
11
treatment. However, prompt identification of the etiological agent along with targeted therapy
12
may result in favourable outcome.
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Bacterial keratitis
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The major bacterial agents of infectious keratitis include Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia and Serratia species (Wong et al., 2012).
16
The community acquired cases of bacterial keratitis are usually resolved with an empirical
17
treatment, however if left un-attempted, may result in perforation, endophthalmitis and loss of
18
vision (Callegan et al., 1994; Wong et al., 2012). Clinical presentation of bacterial keratitis
19
include acute pain, redness, photophobia and corneal ulceration (Stapleton et al., 2007).
20
Pseudomonas ulcers are more severe at presentation than other bacterial ulcers and are often
21
difficult to treat, resulting in worse visual outcome than other bacterial ulcers (Sy et al., 2012;
22
Green et al., 2008).
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Bacterial keratitis accounts for approximately 90% of all microbial keratitis cases (Musa et al., 2010) with P. aeruginosa as the most common culprit, worldwide (Dutta et al., 2012).
3
Corneal infection of Pseudomonas is most commonly associated with the use of contact lenses
4
and was rarely reported as a problem prior to the emergence of contact lens (Marquart &
5
Callaghan, 2013; Ramirez et al., 2012). The resistance of Pseudomonas to disinfectants, coupled
6
with its adherence capability to plastics, facilitates its introduction into the eye where it can react
7
with defective corneal epithelium, gaining further entrance into the corneal stroma. S. pneumonia
8
on the other hand is the major cause of corneal ulcers in developing countries however, some
9
reports emphasize on the fact that Streptococcus are most commonly encountered after P.
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aeruginosa and/or S. aureus eye infections (Bharathi et al., 2010; Yilmaz et al., 2007; Marquart
11
& Callaghan, 2013). Unlike P. aeruginosa, pneumococcal keratitis is not commonly associated
12
with the use of contact lenses and the predisposing factors often include ocular trauma or surgery
13
(Marquart and Callaghan, 2013; Wagoner et al., 2007; Cosar et al., 2001; Lifshitz et al., 2005;
14
Mulet et al., 2010; Rehany et al., 2004).
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Along with P. aeruginosa, S. aureus is also a common etiological agent (Marquart & Callaghan, 2013; Liesegang, 1998; Wong et al., 2011; Jeng et al., 2010; Palmer & Hyndiuk,
17
1993). It is a commensal organism that can readily gain access into the eye, given the
18
opportunity (Mohammadinia et al., 2012; Tacconelli & Johnson, 2011; Speaker et al., 1991).
19
Individuals whose eyes are compromised due to various reasons including ocular surgery or
20
trauma, contact lens use, viral infection or other eye illnesses are at high risk of developing this
21
infection (Cheung & Slomovic, 1995; Ormerod et al., 1987; Coster & Badenoch, 1987). In
22
particular, the ability of S. aureus to develop antibiotic resistance makes this infection among the
23
most difficult one to treat (Asbell et al., 2008). This, along with Serratia marcescens, which once
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was considered a harmless saprophyte, has now been increasingly isolated from the corneal
2
surfaces of keratitis patients (Parment, 1997). The isolation rate of S. marcescens from contact
3
lens-coupled corneal infections ranges from 5 – 28%; almost comparable to the isolation rate of
4
P. aeruginosa in contact lens-related keratitis (Schein et al., 1989; Hume et al., 2007). S.
5
marcescens is a motile gram negative rod abundantly dispersed in nature and contributes to
6
contact lens-related keratitis (Parment et al., 1996; Parment, 1997).
7
4.1 Mechanism
8
4.1.1
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Adhesion
Bacteria initiate infection by engaging with the host cell-surface receptors via various
10
adhesins, which mediate bacterial binding to corneal epithelial cells. Microbial adhesins not only
11
play a role in bacterial attachment to the surface of epithelial cells but may also play an active
12
role in subsequent interactions and infective process. They may act as toxins initiating microbial
13
invasion and contribute to subsequent pathogenic cascade. Bacteria display several adhesins on
14
their surface such as pili or fimbriae, which recognize specific carbohydrates or proteins on the
15
surface of host cell. The adherence of P. aeruginosa, S. pneumonia and S. aureus is significantly
16
higher to damaged corneal epithelium compared to other bacteria accounting for their frequent
17
isolation from keratitis cases.
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Studies have shown that purified pili successfully compete with cold bacteria for binding to
19
ocular surface and have been used to protect against P. aeruginosa keratitis. Corneal epithelial
20
glycoproteins act as surface receptor mediating pilus binding activity and amino sugar sialic acid
21
was able to completely inhibit pilus binding to mouse corneal epithelial cells (Hazlett et al.,
24
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1992; Rudner et al., 1992). Bacterial flagella on the other hand are filamentous organelles,
2
responsible for bacterial motility and therefore are also responsible for dissemination of
3
infection. More than 95% of P. aeruginosa clinical isolates are flagellated and flagella-deficient
4
mutants have been observed to be non-virulent (Ansorg et al., 1984). In addition the anti-
5
flagellar antibody homologous to the infecting strain protects mice from Pseudomonas corneal
6
infection (Rudner et al., 1992). Furthermore, the glycocalyx may also play an important role in
7
bacterial adhesion by producing slime aggregates resistant to phagocytosis thereby enabling
8
them to adhere to cells (Hyndiuk, 1981).
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S. aureus surface adhesins, collectively known as MSCRAMMS (microbial surface components recognizing adhesive matrix molecules), have also been recognized to mediate
11
bacterial adherence to host extracellular matrix components, collagen, fibronectin, fibrinogen,
12
laminin and elastin (Jett & Gilmore, 2002). Rhem et al., (2000), studied the role of MSCRAMM
13
Cna (collagen-binding adhesin) in S. aureus keratitis and concluded that collagen-binding
14
adhesin is a virulence factor for S. aureus keratitis and is involved in the early events of
15
pathogenesis of S. aureus infection of the cornea. Similarly, Jett & Gilmore (2002) studied the
16
role of S. aureus fibronectin-binding protein as an epithelial and/or endothelial cell
17
adhesin/invasion protein. Their data suggested that FnBPs is a surface ligand for human corneal
18
cells and play a key role in host-parasite interactions. It serves as an important adhesin and
19
triggers invasion in ulcerative keratitis caused by S. aureus.
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20
Likewise, Streptococci colonize different sites on human body by expressing multiple
21
adhesins and hence their attachment to human tissues is mediated by a diverse group of bacterial
22
surface proteins, MSCRAMMS (Hammerschmidt, 2006). Plasmin and fibronectin binding 25
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protein A (PfbA), a MSCRAMMS, is known to promote adherence and invasion of bacteria to
2
human epithelial cells by recognizing molecules like fibronectin. In addition, pneumococcal
3
surface adhesin A (PsaA), pneumococcal surface protein A (PspA), pneumolysin (ply),
4
pneumococcal adherence and virulence factor A (PavA), choline-binding protein A
5
(CbpA/PcpA), putative protease maturation protein A (PpmA), IgAI protease (IgAIp), and the
6
streptococcal lipoprotein rotamase A (SIsA) have all been shown to be associated with
7
pneumococcal adherence and virulence (Rajam et al., 2008). Streptococcal cell surface may
8
contain fibrillar structure like pili and fibrils which may also mediate attachment to cell surface
9
to initiate infection. The pneumococcal surface protein C (PspC) promotes adherence and uptake
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of pneumococci into nasopharyngeal epithelial cells (Hammerschmidt et al., 2000). Also,
11
antibodies against PsaA which code for pneumococcal surface adhesin A (PsaA) resulted in
12
reduced adherence to nasopharyngeal epithelial cells (Romero-Steiner et al., 2003). Therefore,
13
these adhesins are likely involved in the initial stages of Streptococcal ocular infection.
14
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Serratia marcescens on the other hand possess mannose-specific adhesins, associated with flagella. These adhesins probably interact with mannose glycoproteins expressed on the surface
16
of corneal epithelial cells as a result of abrasion or trauma due to the use of contact lenses.
17
Labbate et al., (2007), identified type I fimbriae as the critical adhesin that mediate attachment of
18
S. marcescens to human corneal epithelial cell surface. They also identified two AHL (N-
19
acylhomoserine lactone)-regulated genes, bsmA and bsmB, to be involved in adhesion to biotic
20
surface and the expression of these genes leads to the attachment of S. marcescens to HCE cells.
21
In 2007, Shanks et al., demonstrated the role of oxyR and type I fimbrial genes and concluded
22
that they are involved in cell-cell and cell-biotic surface interactions. In addition, two classes of
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1
pili, mannose-resistant and mannose-sensitive pili may also play a role in the attachment of
2
bacteria to epithelial cells (Hejazi & Falkiner, 1997).
3
4.1.2
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Bacterial invasion and cytotoxic effects Once adhered to the epithelial surface, the pathogen invades into the corneal stroma. This
invasion is facilitated via proteases, exotoxins resulting in degradation of basement membrane
6
and extracellular matrix, causing cells to lyse. A number of exotoxins including heat-stable
7
haemolysin, phospholipases, exotoxins play a role in invasion of bacteria into the cornea with
8
eventual stromal necrosis (O’Brien, 2003). Once bacterial invasion into the cornea has ensued,
9
the infection progresses rapidly towards melting of cornea facilitated by bacterial proteases,
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activation of metalloproteases and stimulation of immune response resulting in further damage
11
via release of reactive oxygen intermediates and host proteases (Thidodeaux et al., 2007;
12
Marquart & Callaghan, 2013). Proteases contribute to the pathogenesis of keratitis by degrading
13
basement membrane, laminin, proteoglycans, collagen, and extracellular matrix (Heck et al.,
14
1986; Twining et al., 1986).
P. aeruginosa is capable of secreting at least 7 different proteases including elastase A and
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B, modified elastase, alkaline protease, protease IV, P. aeruginosa small protease and large
17
exoprotease, some of which play a potential role in the pathogenesis of keratitis (Twining et al.,
18
1993; Marquart et al., 2005). Metalloproteases especially elastase B (Las B) and alkaline
19
protease (AP), play a considerable role in keratitis as Las B injection into the corneal stroma
20
result in significant corneal damage (Ijiri et al., 1993; Kessler et al., 1977). Protease IV (PIV) on
21
the other hand cleaves variety of host defense proteins including immunoglobulins, complement
22
components, antimicrobial peptides and surfactants and hence contributes to the virulence of the
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organism (Engel et al., 1998; Alionte et al., 2001). Pseudomonas aeruginosa small protease
2
(PASP) is able to cleave collagen, the chief structural component of the corneal stroma and
3
hence it could play an important role in the obliteration of cornea (Tang et al., 2009). Purified
4
PASP when injected into the rabbit cornea resulted in the destruction of the epithelium and the
5
formation of erosions that can reach into the stroma. These findings suggest that PIV provides
6
bacteria with a defense arm against multiple host defense molecules, while PASP degrads
7
collagen-based structural component of the eye, thereby mediating the pathophysiology of
8
keratitis.
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Mochizuki et al., (2014) determined the role of MucD protease of P. aeruginosa in
10
keratitis in the cornea of mice. The number of bacteria and clinical score in eyes infected with
11
mucD deficient strain was significantly less compared to the parent or rescued strain. In addition
12
large number of infiltrating PMN cells were observed in mucD deficient eyes along with higher
13
MIP2 levels. It was concluded that MucD protease of P. aeruginosa suppressed IL-1β, KC and
14
MIP2 as well as inhibited neutrophil recruitment in the cornea and hence plays a critical role in
15
P. aeruginosa keratitis by facilitating the evasion of immune response.
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Karthikeyan et al., (2013), examined the expression of three main exotoxins, ExoS, ExoT and ExoU, in clinical isolates of P. aeruginosa and showed that majority of strains (84%) were
18
ExoS+ and ExoT+ but lacked ExoU. However, 5 out of 7 strains that were expressing ExoU
19
were also positive for ExoS which is usually a rare phenotype. These exotoxins are directly
20
injected into the host cell via type III secretion system of P. aeruginosa where they exert their
21
cytotoxic effects onto the host cell (Hauser, 2009). ExoU expressing strains cause rapid cell lysis
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1
whereas, ExoS strains are invasive in nature causing membrane blebbing within epithelial cells
2
which are utilized as a site for bacterial replication and motility (Fleiszig et al., 1996). The capsular polysaccharide of S. pneumoniae which once was considered the central
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dogma in pneumococcal virulence is proven to be untrue in the case of keratitis; as noncapsular
5
strains of S. pneumoniae are also able to cause severe keratitis in rabbit keratitis infection models
6
(Norcross et al., 2010; Reed et al., 2005). Other than capsule, the most studied virulence factor
7
of pneumococcus is pneumolysin, a toxin belonging to the family of bacterial cholesterol-
8
dependent cytolysin. It causes direct cell damage by binding to the cholesterol in the host cell
9
membrane followed by polymerization into 30-50 mers, thereby forming pores in the host cell
10
membranes (Morgan et al., 1995; Tilley et al., 2005). In addition to forming pores, it initiates
11
immune-derived damage by activating complement system and induces inflammation (Paton et
12
al., 1984). Futhermore, reduced corneal virulence was demonstrated by of pneumolysin-deficient
13
strain of S. pneumoniae in a rabbit model of intrastromal infection. It was shown that mutation in
14
the complement activation domain of pneumolysin resulted in reduced polymorphonuclear
15
leukocyte (PMN) recruitment and therefore less corneal damage and virulence compared to the
16
wild-type strain (Johnson et al., 1995).
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Proteins other than pneumolysin, includes choline-binding proteins like pneumococcal
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surface proteins A and C (PspA and PspC), neuraminidase A (NanA) and a variety of other
19
surface-associated proteins such as those involved in adherence, immune evasion, activation and
20
enzymatic reactions have been identified to be involved in non-ocular models of infection
21
Recently a metalloprotease, ZmpC, has been shown to induce ectodomain shedding of the
22
membrane-associated mucin (MAM) from conjunctival and corneal epithelial cells leading to the 29
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loss of glycocalyx barrier function and enhanced internalization of bacterium. It was concluded
2
that the removal of MAMs may serve to be an important virulence mechanism employed by S.
3
pneumoniae (Govindarajan et al., 2012).
4
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The alpha-toxin is a pore-forming lytic cytotoxin produced by nearly all strains of S. aureus. Once it gains entry into the cytoplasmic membrane, it moves laterally until seven
6
subunits combine in a circular arrangement (Menestrina et al., 2001; Pany et al., 2004;
7
Vijayvargia et al., 2004). The individual toxin molecules interact with cavoline-1, thereby
8
forming pores in the membrane. Callegan et al., (1994) studied the role of alpha-toxin in the
9
corneal virulence of S. aureus and concluded that it plays a major role in staphylococcal keratitis,
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causing destruction of corneal tissues in the infected eye. Later, Moreau et al., (1997) showed
11
that even nanogram quantity of purified alpha-toxin causes extensive sloughing of the rabbit
12
corneal epithelium, corneal edema and severe iritis. Gamma-toxin, a two component toxin from
13
S. aureus is composed of an F component and an S component that are non-toxic on their own.
14
The S component binds to the target cell followed by binding of the F component both of which
15
then move laterally in the cell membrane joining the other F-S pairs to form a ring that penetrates
16
the membrane resulting in cell lysis (Gravet et al., 1998; Szmigieski et al., 1999). The
17
pathogenic role of gamma- and alpha-toxin were determined by Dajcs et al., (2002) who
18
illustrated that the virulence of strain, Newman is mediated by both gamma- and alpha-toxin,
19
with alpha-toxin mediating corneal epithelial erosions. The strain deficient in either toxins were
20
considerably less virulent compared to parent or rescued strains. S. aureus secretes several two
21
component toxins each of which has its own S and F components (Gravet et al., 1998). This two
22
component toxin system of S. aureus is complicated by the fact that S component of one toxin
23
can also bind to the F component of the other toxin thereby creating several unique combinations
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with their own specific toxicity. The role of these toxins in corneal virulence is yet to be
2
determined. The protein produced by setnm-1 gene has been shown to cause extensive corneal
3
damage; an attribute mediated by its protease activity and is now considered to be important in
4
the virulence of S. aureus keratitis (Caballero et al., 2008). In addition, mutant deficient in this
5
gene is considerably less virulent compared to its parent or rescued strain (Caballero et al.,
6
2010).
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Similarly, intra-corneal injections of sub-microgram amounts of Serratia metalloprotease were able to elicit a rapid and extensive corneal tissue damage of rabbit corneas by causing
9
liquefactive necrosis and descemetocele formation (Kreger & Griffin, 1975; Lyerly & Kreger,
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1979). Lyerly et al., (1981) showed that acute inflammation, liquefactive necrosis of rabbit
11
cornea and descemetocele formation occurred after intra-corneal injection of Serratia protease.
12
In addition, they also showed the in vitro solubilization of the stromal proteoglycan ground
13
substance. Ultramicroscopic examination of damaged cornea revealed loss of ruthenium red
14
staining of the proteoglycan ground substance and dispersal of ultra-structurally normal collagen
15
fibrils. Therefore, the major corneal damage after the injection of Serratia protease is due to the
16
solubilization and loss of ground substance of the tissue and the proteases are at least in part
17
involved in the production of severe corneal damage caused by this bacterium. Kamata et al.,
18
(1985) identified a 56-kDa protease as one of the major factors contributing to the pathogenesis
19
and tissue destruction caused by this bacterium. Vaccination of rabbits with purified protease
20
preparations from S. marcescens exhibited significantly less corneal destruction upon corneal
21
encounter with live bacteria, indicating that proteases are important virulence factors during the
22
development of serratial keratitis (Kreger et al., 1986). Marty et al., (2002) showed that mutant
23
strains of S. marcescens that are deficient in the production of 56 kDa metalloprotease are
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considerably less cytotoxic to mammalian cells. It was further revealed that the culture filtrates
2
of wild-type bacteria pre-treated with EDTA or 1,10-phenanthroline (inhibitor of
3
metalloproteases) exhibited significantly reduced cytotoxicity.
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Gram negative lipopolysaccharide is also an important virulence factor in infectious
5
keratitis. It contributes to the pathogenesis of keratitis by mediating the attachment to the cornea
6
and contact lenses, bacterial internalization and survival inside corneal epithelial cells. In
7
addition, it confers resistance to the complement-mediated killing and stimulates neutrophil
8
migration and infiltration into the cornea with subsequent corneal scarring and opacification.
9
Also, exopolysaccharide formation by both gram positive and gram negative bacteria results in
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local immunosuppressive effects as well as interference with phagocytosis.
11
4.1.3
Stromal necrosis and production of ring infiltrate
Bacterial exotoxins and proteases are constitutively released during multiplication of
13
bacteria. These toxins and enzymes persist in the cornea for a prolonged period of time causing
14
continual stromal destruction and can deprive eye of its vision. Most exotoxins are heat-labile
15
and have antigenic properties. The LPS, an endotoxin within the cell wall of gram negative
16
bacteria is released resulting in the production of stromal rings. These rings consist of
17
polymorphonuclear leucocytes within the corneal stroma, which are chemo-attracted by the
18
alternate complement pathway.
19
4.2 Treatment
20 21
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Due to the rapid progression of bacterial keratitis, it should be treated as a medical emergency and empirical antibiotic treatment should be promptly commenced. The objective for 32
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the initial therapy is the rapid eradication of the corneal pathogen. Basically, no single antibiotic
2
is effective against all bacterial keratitis-causing organism and therefore an agent having broad
3
spectrum activity covering both, gram negative and gram positive organisms is desirable.
4
Broadly speaking, two treatment options are available; fluoroquinolones monotherapy and/or
5
combination therapy of fortified antibiotics including cefazolin and tobramycin or gentamicin
6
(Gokhale, 2008). The aminoglycosides in fortified drops provides excellent coverage against
7
gram-negative organisms and are also active against Staphylococci and some Streptococci.
8
Cephalosporin on the other hand gives good coverage for non-penicillinase producing gram-
9
positive bacteria. Fluoroquinolone antibiotics have a broad spectrum activity against both gram-
10
negative and gram-positive organisms including penicillinase-producing and methicillin resistant
11
Staphylococci. They inhibit DNA gyrase and topoisomerase IV; the key enzymes involved in
12
DNA replication and transcription leading to bactericidal effect of these antibiotics (Wong et al.,
13
2012; Pestova et al., 2000; Blondeau, 2004).
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The frequency of application of antibiotic drops depends on the severity of infection but usually they are administered every half an hour for the first 24 – 36 hours (O’Brien, 2003;
16
Gokhale, 2008). In severe cases, an initial loading dose is achieved via a drop every 5 min for the
17
first 30 min. The concept behind the loading dose is the vascularity of the cornea and poor
18
penetration of the drug into the corneal stroma which requires frequent topical applications to
19
achieve minimal inhibitory concentration. The therapy is then tapered off gradually based on the
20
clinical response of the patient. Other antibiotics used include amikacin, vancomycin,
21
methicillin, clotrimoxazole, and clarithromycin depending on the causative agent responsible for
22
keratitis (Gokhale, 2008).
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Owing to the rich innervation of the cornea, the disease is frequently associated with
2
considerable pain and therefore analgesics or pain control medications are recommended, which
3
result in improved patient comfort and effective delivery of the treatment regimen. The use of
4
corticosteroids as anti-inflammatory agents are controversial and are best avoided until the
5
infection is completely eradicated or at least under control (O’Brien, 2003; Gokhale, 2008). In
6
patients with corneal ulceration the use of non-steroidal anti-inflammatory drugs should also be
7
avoided due to increased risk of corneal melting and perforation. Topical cycloplegics are used
8
to relieve ciliary spasm, pain and formation of synechiae. Secondary glaucoma may result in
9
keratitis due to increased intraocular inflammation and hence must be treated with topical
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antiglaucoma medications.
In the case of marked corneal ulceration, the temporary use of therapeutic soft contact
12
lenses may facilitate stromal repair and promote re-epithelialization by protecting corneal surface
13
from mechanical trauma. They may act as a tear film antibiotic retention device thereby
14
facilitating penetration by prolonging contact time (Matoba & McCulley, 1985; Busin &
15
Spitznas, 1988). In addition, collagen corneal shields soaked in antibiotic solution have also been
16
used as an effective adjunct and have shown to increase antibiotic penetration (O’Brien et al.,
17
1988; Unterman et al., 1988). Furthermore, temporary intracanalicular collagen implants and
18
liposomal systems have also been designed to prolong drug retention and interaction (Gilbert et
19
al., 1986; Schaeffer & Krohn, 1982).
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Therapy for bacterial keratitis is not only to eradicate the causative agent but also to
21
prevent tissue destruction and irreversible structural alterations caused by enzymes and toxins
22
released by the bacteria. Enzyme inhibitors like disodium edetate, acetylcysteine, Heparin have 34
ACCEPTED MANUSCRIPT
been shown to be effective experimentally (Slansky et al., 1971; Ellison & Poirier, 1976; Mehra
2
et al., 1975). Synthetic inhibitors of matrix metalloproteases have been shown to inhibit P.
3
aeruginosa proteases in experimental Pseudomonas keratitis (Barletta et al., 1996). The corneal
4
stroma which constitutes about 90% of corneal thickness is made up of regularly arranged
5
collagen fibrils. Thus the collagenases from bacteria, digest the human collagen causing the
6
cornea to melt. Moreover, collagen cross linking is a useful technique which uses riboflavin and
7
UVA irradiation to strengthen corneal tissues thereby enhancing its rigidity (Spoerl et al., 1998;
8
Spoerl & Seiler, 1999; Wollensak et al., 2003). The interaction between riboflavin and UVA
9
strengthens chemical bond formation between collagen fibrils and hence increasing its resistance
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to enzymatic digestion (Spoerl et al., 2004). In addition, the photoactivation of riboflavin has
11
cidal effect on microorganisms by causing damage to microbial DNA and/or RNA (Wong et al.,
12
2012).
In case of progressive corneal thinning or perforations, less than 2 mm cyanoacrylate tissue
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adhesives may be used, which may also have some inherit antibacterial activity as well
15
(Eiferman & Snyder, 1983; Gokhale, 2008; O’Brien, 2003; Kirkness et al., 1991). Tissue
16
adhesives are only suitable for small perforations, as they are toxic to corneal endothelium. In
17
case of large perforations, therapeutic penetrating keratoplasty may be required. Corneal patch
18
grafting may be an alternative to tissue adhesives and conjunctival flaps may help selected cases
19
of refractory ulceration to assist healing. Severe bacterial keratitis may also result in cataract
20
formation due to bacterial toxins, iridocyclitis and treatment toxicity (Lotti & Dart, 1992).
21
Surgical intervention may be required in such cases depending on the degree of corneal scarring
22
and opacification.
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1
5
Concluding remarks Microbial keratitis, is a complex entity with many considerations when it comes to its
3
treatment. It is a major public health concern particularly in developing countries where access to
4
care is limited and economic barriers are huge where it can become a leading cause of visual loss
5
in a population that is young. As with all corneal infections, proper identification of the causative
6
agent followed by appropriate targeted therapy can abate the risk of complications. A better
7
understanding of the pathogenic cascade would no doubt lead to improved clinical treatment.
8
However the emergence of drug-resistant strains along with the recurrence of infections
9
emphasise on the need for more effective therapeutic modalities. Additionally, the need to
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identify the genetic basis of virulence factors responsible for the disease is also important, as the
11
pathogenicity of keratitis is a sum of multiple events occurring together in time and space for the
12
successful transmission of the pathogen to the susceptible host, overcoming its physiological
13
barriers and eventually producing disease. Subsequently, a complete understanding of
14
pathogenesis of the particular organism will undoubtly lead to the identification of potential
15
therapeutic interventions.
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Acknowledgments: This work was supported by Aga Khan University, Pakistan and Sunway
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University, Malaysia.
64
Conflict of Interests: The authors declare that there is no conflict of interests regarding the
65
publication of this paper.
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Tronchin G, Pihet M, Lopes-Bezerra LM, Bouchara JP (2008): Adherence mechanisms in human
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Vemuganti GK, Garg P, Gopinathan U, Naduvilath TJ, John RK, Buddi R, Rao GN (2002): Evaluation of agent and host factors in progression of mycotic keratitis: A histologic and
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Xie L, Zhai H, Zhao J, Sun S, Shi W, Dong X (2001): Antifungal susceptibility for common pathogens of fungal keratitis in Shandong Province, China. Am J Ophthalmol 146: 260-265.
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Xie L, Zhong W, Shi W, Sun S (2006): Spectrum of fungal keratitis in north China. Ophthalmol 113: 1943-1948.
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Yilmaz S, Ozturk I, Maden A (2007): Microbial keratitis in West Anatolia, Turkey: a retrospective review. Int Ophthalmol 27: 261-268. Yoon KC, Jeong IY, Im SK, Chae HJ, Yang SY (2007): Therapeutic effect of intracameral
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amphotericin B injection in the treatment of fungal keratitis. Cornea 26: 814-818.
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ACCEPTED MANUSCRIPT Highlights Microbial keratitis is caused by bacteria, fungi, and protist pathogens
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Epithelial defects and injuries are key predisposing factors in microbial keratitis
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Factors from the host and the pathogen contribute to the disease.
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Successful prognosis requires early diagnosis and aggressive treatment.
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This is a timely review of our current understanding of microbial keratitis.
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S0882-4010(16)30758-6
DOI:
10.1016/j.micpath.2016.12.013
Reference:
YMPAT 2034
To appear in:
Microbial Pathogenesis
Received Date: 8 November 2016 Revised Date:
30 November 2016
Accepted Date: 5 December 2016
Please cite this article as: Lakhundi S, Siddiqui R, Khan NA, Pathogenesis of microbial keratitis, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2016.12.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Pathogenesis of microbial keratitis
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Sahreena Lakhundi, 2Ruqaiyyah Siddiqui, 2Naveed Ahmed Khan*
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Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan;
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Department of Biological Sciences, Faculty of Science and Technology, Sunway University,
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Selangor, Malaysia.
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Short title: Microbial keratitis
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*Corresponding address: Department of Biological Sciences, Faculty of Science and
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Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext: 7176.
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Fax: 60-(0)3-5635-8630. E-mail: [email protected]
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Abstract Microbial keratitis is a sight-threatening ocular infection caused by bacteria, fungi, and protist pathogens. Epithelial defects and injuries are key predisposing factors making the eye
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susceptible to corneal pathogens. Among bacterial pathogens, the most common agents
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responsible for keratitis include Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus
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pneumonia and Serratia species. Fungal agents of corneal infections include both filamentous as
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well as yeast, including Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia, Paecilomyces,
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Scedosporium and Candida species, while in protists, Acanthamoeba spp. are responsible for
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causing ocular disease. Clinical features include redness, pain, tearing, blur vision and
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inflammation but symptoms vary depending on the causative agent. The underlying molecular
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mechanisms associated with microbial pathogenesis include virulence factors as well as the host
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factors that aid in the progression of keratitis, resulting in damage to the ocular tissue. The
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treatment therefore should focus not only on the elimination of the culprit but also on the
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neutralization of virulence factors to minimize the damage, in addition to repairing the damaged
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tissue. A complete understanding of the pathogenesis of microbial keratitis will lead to the
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rational development of therapeutic interventions. This is a timely review of our current
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understanding of the advances made in this field in a comprehensible manner. Coupled with the
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recently available genome sequence information and high throughput genomics technology, and
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the availability of innovative approaches, this will stimulate interest in this field.
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Key words: Cornea; Keratitis; Protist; Bacteria; Yeast
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1. Introduction The unique structure of the human eye along with its exposure to environment, renders it susceptible to a number of agents responsible for causing infection. Injuries and epithelial defects
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impair defense mechanisms and exposure to pathogenic microbes can lead to corneal
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inflammation or keratitis. The intact ocular surface thwarts most microorganisms but once
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anatomical barriers are breached, host defenses against pathogens are less than sufficient to
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prevent infection that can lead to eventual loss of vision. Microbial or infectious keratitis is a
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potentially sight-threatening ocular condition caused by bacteria, fungi, protists etc. It is the
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inflammation of the cornea caused by pathogenic microbes that eventually invades the corneal
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stroma causing inflammation, and ultimately destruction of these structures (Ansari et al., 2013;
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Wong et al., 2012). The most common pre-disposing factors to develop infectious keratitis
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include the use of contact lenses, especially overnight or extended wear lenses, inadequate
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disinfecting solutions, trauma, ocular surgery especially corneal surgery, chronic ocular surface
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disease, systemic disease like diabetes mellitus and/or extended use of topical corticosteroids
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(Stapleton et al., 2012; Wong et al., 2012). Patients usually present with redness, tearing, rapid
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onset of pain and blur vision. Clinical presentation may vary depending on the causative agent
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responsible for causing keratitis. The condition should be treated as a medical emergency and
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adequate treatment should commence promptly. If appropriate antimicrobial treatment is
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delayed, only 50% of the eye gains good visual acuity (Jones, 1981). Appropriate management
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and timely onset of treatment can reduce the incidence of severe visual loss restricting corneal
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damage. Here, we present a concise review of the bacterial, fungal and protist keratitis.
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Numerous microorganisms can infect the eye either by direct or indirect introduction into the
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eye. The most common clinically important microorganisms involved in eye infections are
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reviewed in this article with relation to the anatomical part of the eye involved in the disease,
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along with a discussion of the pathogenic mechanisms and management of the disease.
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2. Acanthamoeba keratitis
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Acanthamoeba keratitis is a rare but sight threatening corneal infection, caused by an
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opportunistic protist pathogen belonging to the genus Acanthamoeba. They are ubiquitous, free-
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living protists dispersed in a variety of environments including air, soil, freshwater, tap water,
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hospital equipments, surgical instruments, showers, ventilation ducts, air-conditioning units,
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chlorinated swimming pools, sewage etc. (De Jonckheere, 1991; Kilvington & White, 1994).
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Phenotypic switching into a cyst form enables Acanthamoeba to withstand adverse
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environmental conditions. The Acanthamoeba trophozoite has an amoeboid shape that contains
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spike-like structures known as acanthopodia and during this stage, amoebae feed and reproduce
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under favourable environmental conditions (Marciano-Cabral & Cabral, 2003; Visvesvara et al.,
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2007). However, under extreme situations such as lack of nutrients, hyperosmolarity,
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desiccation, extreme pH, temperatures, and the presence of antimicrobials; the trophozoite
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rounds up and confines itself within a double-walled resistant cyst form that has minimal
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metabolic activity. The cyst stage presents a major problem in the successful treatment of
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Acanthamoeba infections as they are resistant to various antimicrobial agents, often leading to
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recurrence of the disease upon discontinuation of therapy (Niederkorn et al., 1999). Although
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exposure to Acanthamoeba spp. appears to be common due to its ubiquitous nature, the incidence
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of Acanthamoeba keratitis is less common. The main risk factors for Acanthamoeba keratitis are
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contact lens wear for extended periods, corneal trauma, non-sterile contact lens rinsing,
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swimming while wearing contact lenses and biofilm formation on contact lens (Marciano-Cabral
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& Cabral, 2003; Trabelsi et al., 2012). While contact lens wear is the leading risk factor,
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Acanthamoeba can cause keratitis in non-contact lens wearers as well (Dart et al., 2009).
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However, it is often overlooked in non-CLs users as a causative agent of keratitis, where it is
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usually associated with trauma and/or exposure to contaminated water, soil and organic matter.
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In addition, Acanthamoeba keratitis has been reported after invasive or radial keratoplasty and/or
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laser-assisted in situ keratomileusis (LASIK) where the lesions generally worsen due to a delay
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in the diagnosis and treatment (Garg et al., 2010; Sharma et al., 2011).
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Clinical manifestations of Acanthamoeba keratitis include excruciating pain characterized
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by redness, epiphora, lacrimation, eyelid diptosis, conjonctival hyperhemia, foreign body sensation and photophobia (Niederkorn et al., 1999; Mahgoub, 2010; Alizadeh et al., 1994). As
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the disease progresses, stromal involvement results in infiltration of inflammatory cells
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displaying a characteristic ring infiltrate. If not promptly diagnosed and treated aggressively,
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cornea becomes ulcerated leading to perforation, ring infiltrate, stromal abscess formation, loss
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of visual acuity and eventually blindness and enucleation. Acanthamoeba was first recognized as an ocular pathogen in 1973 in the U.S.A where the
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first case was reported by an ocular microbiology group in Dallas (Clarke et al., 2012). However,
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the first published report of Acanthamoeba keratitis emerged in the UK in 1974 and was
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associated with minor eye injury (Naginton et al., 1974). The first case in a contact lens wearer
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was reported 10 years later in 1984 from a patient wearing soft contact lenses (Samples et al.,
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1984). Since then the number of cases has been on the rise especially in recent years, mainly due
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to an increase in the number of contact lens users, better diagnostics and increased awareness.
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2.1.
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2.1.1. Adhesion The first step in the pathogenesis of Acanthamoeba keratitis is the ability of amoebae to
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bind to the corneal epithelium, a factor that determines the degree of pathogenicity of different
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isolates. The pathogenic cascade begins via amoebal binding to mannose glycoproteins on the
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corneal surface through adhesin expressed on the trophozoite membrane called the mannose-
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binding protein (MBP) (Panjwani, 2010; Clarke & Niederkorn, 2006; Garate et al., 2004). This
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adherence is a crucial prerequisite for producing infection. In addition. mild trauma or corneal
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abrasion is required for the establishment of corneal infection. Abrasion or trauma results in
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increased expression of mannose glycoproteins on the corneal epithelium and hence there is
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increased adhesion of amoebae to the damaged cornea, compared with the healthy cornea
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(Clarke & Niederkorn, 2006). The contact lenses, in addition to serving as a vector for the
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introduction of trophozoite onto the corneal surface, also up regulate mannose glycoproteins on
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the corneal epithelium. This results in an increased number of trophozoites binding to the contact
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lens-conditioned cornea compared to the normal cornea thus leading to findings that more than
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80% of Acanthamoeba keratitis cases are associated with the use of CLs (Alizadeh et al., 2005).
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The second important element involved in adhesion is the number of acanthopodia on the
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amoebic surface. Pathogenic amoebae have more than 100 acanthopodia/cell compared to non-
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pathogenic amoebae. Therefore, non-pathogenic amoeba present very low binding levels to host
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cells compared to pathogenic amoebae (Khan, 2001; Panjwani, 2010). Hence the number of
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acanthopodia is also believed to be closely related to the rate of adhesion to the corneal surface.
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Once attached, intracellular signaling processes trigger the pathogenic cascade involved in the
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pathogenesis of Acanthamoeba keratitis.
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2.1.2. Cytopathic effect
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Trophozoite binding to corneal surface is tailed by extensive desquamation of the corneal epithelium, leading to penetration of the underlying Bowman’s membrane. Trophozoite-
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mediated cytopathic effect proceed via several mechanisms such as direct cytolysis, phagocytosis
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and apoptosis (Alizadeh et al., 1999). It has been observed that Acanthamoeba-mediated direct
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cytolysis is dependent on calcium channel activity and on cytoskeletal elements, as the inhibition
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of both via the calcium channel blocker, Bepridil and actin polymerization inhibitor, cytochalasin
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D overcomes Acanthamoeba-mediated cytolysis by over 98% (Taylor et al., 1995). In addition
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the presence of structures like food cups or amoebastomes on the surface of Acanthamoeba
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suggests that the amoeba binding to host cells leads to secondary events like phagocytosis by
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which the amoeba bites or engulfs host cells. It has been observed that Acanthamoeba
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phagocytose and/or engulfs corneal epithelial cells and that this activity is mediated via
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amoebastomes present on the surface of amoebae (Khan, 2001).
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Apart from inducing direct cell death, several studies indicate that Acanthamoeba induces apoptosis of keratocytes, iris ciliary body cells, retinal pigment epithelial cells, corneal epithelial
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cells, neuroblastoma cells etc. (Marciano-Cabral & Cabral, 2003; Niederkorn et al., 1999; Clarke
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et al., 2005; Stopak et al., 1991). Acanthamoeba induces membrane blebbing, formation of
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apoptotic bodies, DNA laddering, nuclear chromatin condensation of host cell, all known
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markers for apoptosis (Alizadeh et al., 1994; 1994a; Pettit et al., 1996). It has been observed that
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exposure to mannose stimulates the production of a 133-kDa protease termed mannose-induced
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protein (MIP133) by Acanthamoeba trophozoites. This production of MIP133 appears to be
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another vital element of the pathogenic cascade of Acanthamoeba keratitis. It initiates apoptosis
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of corneal epithelial cells in a caspase-3-dependent pathway in vitro and it is possible that the
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trophozoite uses this pathway in the desquamation of the corneal epithelial cells in vivo (Hurt et
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al., 2003a; Hurt et al., 2003a). Furthermore, clinical isolates of Acanthamoeba but not soil
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isolates produce MIP133 and can cause disease in animals, signifying an association between
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MIP133 production and pathogenic potential. Hence the trophozoite-mediated cytopathic effect
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to destroy epithelial cells occurs by three independent mechanisms i.e., direct cytolysis,
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phagocytosis and apoptosis.
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2.1.3. Stromal invasion
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Following binding and desquamation of the corneal epithelium, the next step in Acanthamoeba keratitis involves the penetration and dissolution of the underlying collagenous
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stroma. The Acanthamoeba trophozoites secrete a variety of proteases with nonspecific
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collagenolytic activity to facilitate the invasion and degradation of stroma. These include serine
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proteases, cysteine proteases, metalloproteinases, elastase, collagenolytic enzyme, phospholipase
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A and a novel plasminogen activator (Cao et al., 1998; Cho et al., 2000; Hadas & Mazur, 1993).
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Studies have shown the direct role of extracellular proteases in Acanthamoeba keratitis by co-
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incubating corneal epithelial cells with Acanthamoeba conditioned medium, which resulted in
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host cell cytotoxicity (He et al., 1990). This cytotoxic effect was abolished in the presence of a
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serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), suggesting a crucial role played
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by serine proteases in the pathogenicity of Acanthamoeba keratitis (Dudley et al., 2008).
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Furthermore, pathogenic Acanthamoeba exhibit higher protease activity compared to non-
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pathogenic amoebae (Khan et al., 2000). Moreover, serine proteases are also able to degrade
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collagen, fibronectin, laminin, secretory IgA, IgG, plasminogen, BSA, fibrin, fibrinogen, and
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rabbit corneal proteins (Siddiqui & Khan, 2012). These properties of proteases are important
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because collagen type I is the main structural protein in the corneal stroma which is important for
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the maintenance of its integrity. In addition sIgA, the main immunoglobulin in tears and a
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primary barrier against pathogenic microbes are also degraded by these proteases. Other
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extracellular enzymes like elastase and phospholipase A are capable of degrading connective
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tissues. These findings suggest that collagenolytic enzymes have a role in corneal lesions and the
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generation of ring-like stromal infiltrates which are characteristic features of Acanthamoeba
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keratitis (He et al., 1990).
Pathogenic Acanthamoeba can also use plasminogen activator that is constitutively present in ocular isolates, to catalyze the cleavage of host plasminogen to plasmin, which in turn
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activates matrix metalloproteinases (MMPs) to degrade components of extracellular matrix
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(Mitra et al., 1995; Berman, 1993). They result in ulceration of normally quiescent cornea by
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degrading the components of basement membrane and extracellular matrix, including type I and
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II collagens, laminin and fibronectin (Parks, 1999). Corneal stroma, unlike other extracellular
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matrices is vital for the normal functioning of the eye. Its degradation affects normal vision and
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can lead to blindness. Studies have also shown that pathogenic isolates of Acanthamoeba are
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able to secrete ecto-ATPases which play a role in protecting the cells from effector cells of the
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immune system. Secondly, they play a role in repolarization of the membrane after
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depolarization by an active compound by acting as a proton pump. Lastly, these enzymes are
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thought to be related to adherence to the host cell (Siddiqui & Khan 2012).
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2.1.4. Neuritis Acanthamoeba spp. are highly motile and secrete a plethora of proteases that augment their
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pathogenesis by desquamating corneal epithelium, penetrating Bowman’s membrane and
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invading the corneal stroma. This cascade of events comes to a halt and trophozoites almost are
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never able to penetrate the corneal endothelium and the anterior chamber of the eye (Clarke &
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Niederkorn, 2006). However, they seem to cluster around the corneal nerves producing radial
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keratoneuritis (Alizadeh et al., 2005). Studies have demonstrated the chemotactic response of
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Acanthamoeba trophozoites towards extracts of neuronal cells and neural-crest-derived cells but
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not towards the corneal epithelial or stromal cells (Pidherney et al., 1993). Moreover, in vitro
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studies have also demonstrated the killing of nerve cells by trophozoites via direct cytolysis
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and/or apoptosis (Pettit et al., 1996). These cytopathic effects contribute to nerve damage and
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may account for the excruciating pain experienced by the patient, coupled with this infection in
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vivo.
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2.2.
Currently the treatment for Acanthamoeba keratitis involves topical application of cocktail
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of drugs, as there is no single effective drug treatment against Acanthamoeba keratitis. This is
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mainly because of the degree of virulence of the infecting isolate which is different for each
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individual case and therefore makes it impossible to establish a correlation between in vitro and
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in vivo efficacies. The best therapeutic outcomes are expected only when, it is diagnosed early,
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treated adequately and aggressively with a high level of patient compliance. Acanthamoeba
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trophozoites are sensitive to most available drugs; however the cysts present a major issue in the
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course of treatment and are the main reason for the recurrence of infection upon discontinuation
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of therapy. Thus, successful management most often necessitates extended therapy with a
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combination of well tolerated drugs with minimal toxicity to hosts cells that results in a
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favourable visual consequences with reduced need for surgical interventions.
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The most effective topical agents currently used against Acanthamoeba trophozoites and cysts are the biguanides (polyhexamethylene biguanide (PHMB) 0.02 – 0.06% or chlorhexidine
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0.02 – 0.2%) in combination with diamidine (propamidine isethionate 0.1% or hexamidine 0.1%)
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(Wright et al., 1985; Khan, 2003; Dart et al., 2009; Lorenzo-Morales et al., 2013). The former
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drugs are membrane-acting cationic biocides that interact with negatively charged surface
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proteins of Acanthamoeba resulting in leakage of the cellular components (Perrine et al., 1995).
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The latter however exert their amoebicidal effects via cationic surface-active properties inducing
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structural changes resulting in cell permeability and are effective DNA synthesis inhibitors
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(Duguid et al., 1997). In vitro studies revealed that cationic agents have the best and most
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constant amoebicidal and cysticidal activity (Elder et al., 1994; Hay et al., 1994). If these drugs
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are applied early on in the development of infection, at high frequency along with neomycin and
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0.15% dibromopropamidine, they show successful prognosis (Lorenzo-Morales et al., 2013).
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Hourly drops of PHMB and hexamidine day and night may be tapered off after 48 hours to
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alleviate epithelial toxicity, to hourly drops during the day for the next 72 hours. This therapy
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reduces viable trophozoites when they are more susceptible and prevent them from turning into
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fully mature cysts. This treatment is then reduced to 2-hourly application during the day for the
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next 3 – 4 weeks and then gradually tailored depending on the individual case. Treatment
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regimes on average last over a period of 6 months, ranging from 0.5 to 29 months (Radford et
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al., 1998).
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In the case of inflammation along with persistent infection, corticosteroids have been implicated however, there use is controversial as they cause suppression of host immune
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response (Lorenzo-Morales et al., 2013). Recent reports also point towards the use of the azoles
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as an adjuvant to biguanide and diamidine therapy in resistant Acanthamoeba keratitis cases
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(Bang, et al., 2010). Therefore topical and intrastromal voriconalzole drops (1%) have been used
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successfully in 3 cases with resistant Acanthamoeba keratitis. Surgical management via epithelial
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debridement, corneal graft surgery (keratoplasty) and/or DALK (deep lamellar keratoplasty) may
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be required in some cases where the corneas are permanently scarred or where the topical and
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oral treatments have failed (Illingworth & Cook, 1998; Chen et al., 2004; Sony et al., 2002;
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Jiang et al., 2006; Parthasanrathy & Tan, 2007). Recently, photorefractive surgery seems to be a
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very promising modality and have been used in four cases of early stage Acanthamoeba keratitis,
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where the patients developed large corneal abscesses in the upper third thickness of stroma
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(Kandori et al., 2010). Another relatively new approach is collagen cross-linking which has
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shown promising results in clinically (Garduno-Vieyra et al., 2011; Khan et al., 2011; Muller et
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al., 2012). It is presumed that collagen probably prevents further tissue damage and prevents
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reproduction of amoebae and hence the patients showed rapid reduction in ocular symptoms and
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ulcer size.
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Moon et al., (2015) recently reported that autophagy inhibitors have anti-amoebic effects
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and if used with low concentrations of PHMB (0.00125%) has very low cytopathic effect on
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human corneal cells and a high cytopathic effect on Acanthamoeba cells. In addition, Aqeel et
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al., (2015) suggested photochemotherapeutic strategy against Acanthamoeba infections and
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showed that mannose-conjugated porphyrin has the potential for targeted photodynamic therapy
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of Acanthamoeba infections. Regardless of significant advances in Acanthamoeba keratitis 12
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treatment over the last decade or so, prevention still seems to be the best option. In addition,
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contact lens wearers must be thoroughly educated on the proper hygiene to handle contact lenses
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to avoid this serious sight-threatening infection.
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3. Mycotic keratitis
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Mycotic keratitis is the fungal infection of the cornea caused by either filamentous and/or
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yeast-like fungi. They may account for more than 50% of all culture-positive microbial keratitis
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especially in tropical and sub-tropical countries (Xie et al., 2006; Nath et al., 2011; Thomas &
8
Kaliamurthy, 2013). A strong geographical correlation has been reported to exist between the
9
occurrences of different types of keratomycosis. For example, the proportion of keratitis due to
10
yeast-like fungi show a tendency to increase towards temperate climates whereas corneal ulcers
11
caused by filamentous fungi appear to be more common towards tropical latitudes (Leck et al.,
12
2002).
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Filamentous fungi such as Fusarium, Aspergillus, Phaeohyphomycetes, Curvularia, Paecilomyces and Scedosporium apiospermum are most commonly associated with keratitis
15
caused by filamentous fungi (Thomas & Kaliamurthy, 2013). However Candida albicans and
16
other Candida species are most common keratitis-causing yeast-like fungi. Former is reported to
17
be due to ocular trauma which is usually the most important predisposing factor in healthy young
18
males engaged in agricultural or other outdoor activities (Nath et al., 2011; Gopinathan et al.,
19
2009; Thomas & Kaliamurthy, 2013). These fungi do not invade the intact cornea and
20
penetration only occurs once the epithelium is abraded. Traumatizing agents of animal origin or
21
vegetative matter, soil or dust particle either directly implant fungal conidia on abraded corneal
22
epithelium for fungal invasion (Nath et al., 2011; Gopinathan et al., 2009; Thomas &
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Kaliamurthy, 2013). The use of corticosteroids, ocular surgery, ocular surface disease and
2
contact lens wear has now also been increasingly identified as a significant risk factor.
3
Conversely, C. albicans and related fungi are only linked to keratitis when there is a pre-existing
4
ocular condition like insufficient tear secretion, defective eye closure, or some systemic illness
5
such as diabetes mellitus or immunosuppression (Thomas, 2003). This form of keratomycosis
6
may also supervene on a pre-existing epithelial defect caused by herpes keratitis or abrasion due
7
to contaminated contact lenses.
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Fungal infections of the cornea need to be promptly addressed to facilitate full recovery. In
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case of filamentous fungal keratitis, clinical manifestations include sudden onset of pain along with photophobia, discharge with reduced vision and opacity on the surface of the cornea
11
suggestive of an ulcer (Ansari et al., 2013). It may involve any part of the cornea and show firm,
12
sometimes dry elevated slough, hyphate lines extending into the normal cornea beyond the edge
13
of the ulcers, multifocal granular or feathery grey-white satellite stromal infiltrates, immune ring,
14
Descemet’s fold and mild iritis (Thomas, 1994; Rosa et al., 1994; Srinivasan, 2004; Thomas &
15
Kaliamurthy, 2013). Although each fungal keratitis exhibits these basic features, they may vary
16
depending on the etiological agent. Severe, chronic filamentous keratitis somewhat resemble
17
bacterial suppuration and may involve the entire cornea. However those due to yeast-like and
18
related fungi may resemble bacterial keratitis with an overlying epithelial defect, discrete
19
infiltrate and slow progression (Sun et al., 2007).
20
3.1 Mechanism
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3.1.1
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Adhesion
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Interaction of pathogenic fungi with host cells is the key factor in the pathogenesis of mycotic keratitis. Adherence of microorganisms to host cells is a pre-requisite for the initiation
3
of the infection. Hence fungal infections of the cornea begin via adhesion of fungi to the
4
damaged cornea. Fungal pathogens display a variety of adhesins that are capable of adhering to
5
various cell types and interact with a variety of host proteins and glycoproteins present in host
6
cells (Hostetter, 1994). The outer fibrillar layer of yeast and filamentous fungal cell wall is
7
composed of mannan or mannoprotein (Imwidthaya, 1995; Thomas, 2003). The adhesive
8
mannoproteins play an essential role in fungal binding to corneal tissues. These lectin-like
9
proteins recognize D-mannose or mannose glycoproteins on the surface of corneal epithelial
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cells. Damage to the cornea results in the upregulation of mannose glycoproteins on the corneal
11
surface (Clarke & Niederkorn, 2006); it may therefore play an important role in the adhesion and
12
pathogenesis of fungal keratitis. Ocular trauma is the most important pre-disposing factor in
13
mycotic keratitis (Nath et al., 2011; Gopinathan et al., 2009; Thomas & Kaliamurthy, 2013); the
14
absence of which prevents fungal penetration into the cornea. Thus, the correlation of ocular
15
trauma with an increased expression of mannose glycoproteins on the corneal surface and
16
presence of adhesive mannoproteins on the surface of fungal cell wall is likely involved in the
17
pathophysiology of fungal keratitis.
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The corneal epithelium also possess other potential fungal binding sites like laminin,
19
fibronectin, collagen etc. which also play a role in keratitis (Thomas, 2003). Tronchin et al.,
20
(1988) demonstrated the importance of an outer fibrillar layer of the germ tube in the process of
21
adhesion to plastics. Bouchara et al., (1990) reported that these major components of the fibrillar
22
cell wall layer of the germ tube act as a receptor for laminin, fibrinogen and mediates its
23
attachment to membranes. The involvement of specific interaction of Aspergillus fumigatus with 15
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finrinogen and its role in cell adhesion has shown that fungal conidia adhere avidly to wells
2
coated with laminin, fibrinogen, collagen and fibronectin substrates (Coulot et al., 1994). This
3
was further supported by Bromley & Donaldson (1996), who showed the ability of fibrinogen
4
and laminin to inhibit the adherence of conidia to pulmonary epithelial cell lines. In addition
5
sialic-acid dependent lectins have also been reported to play an important role in recognition of
6
laminin and fibrinogen by Aspergillus and Penicillium conidia.
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Furthermore, the outer layer of conidia also plays a crucial role in the early stages of
8
infectious process. The surface of resting conidia has been reported to have proteins belonging to
9
the hydrophobin family (Parta et al., 1994; Thau et al., 1994) which are detected in all
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filamentous fungi and thought to play a role in adherence (Scholtmeijer et al., 2001).
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3.1.2
The etiological agent in Fusarium keratitis is able to invade the cornea gaining access to
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Invasiveness and Morphogenesis
the anterior chamber of the eye. There, at the pupillary area, it forms a lens-iris-fungal mass
14
affecting the normal drainage of aqueous humor leading to an increase in the intraocular pressure
15
causing fungal malignant glaucoma (Kuriakose & Thomas, 1991). Initially, malignant glaucoma
16
was thought to occur only in Fusarium keratitis, however recently Jain et al., (2007), reported a
17
case of Aspergillus-induced malignant glaucoma where the uniform shallowing of the anterior
18
chamber was present with raised IOP unresponsive to antiglaucoma measures. Eventually A.
19
flavus was isolated from the anterior chamber of the eye.
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Fungal invasiveness is directly related to the fungal load and inversely proportional to the
21
intensity of inflammatory response (Vemuganti et al., 2002). Therefore, the heavier the load of
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the fungus, the greater the extent of invasion and the smaller the number of inflammatory cells.
2
In early mycotic keratitis, the heavy fungal load with deep tissue penetration may overcome the
3
inflammatory response in corneal tissue, encouraging the progression of the disease. Conversely
4
the inflammatory response in early keratitis may be mild and therefore the fungi multiply
5
extensively and penetrate deep into the tissues. The putative sequence of events in which it
6
occurs still needs to be confirmed.
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Phenotypic switching or morphogenesis is an important method of adaptation used by
8
some microbes in different microenvironments to survive inside infected hosts. This permits
9
fungi to survive in the presence of anti-fungal drugs and resist anti-microbial therapy. The same
10
intrahyphal hyphae or hypha-in-hypha and thickened fungal cell walls are seen in corneal tissues
11
infected with L. theobromae and in corneas infected with F. solani keratitis treated earlier with
12
corticosteroids, as seen in the presence of antifungal drugs (Thomas et al., 1991; Kiryu et al.,
13
1991). This suggests that these morphological alterations may allow fungi to evade host defenses
14
and present a barrier against antifungal drugs. Jackson et al., (2007) demonstrated the role of
15
EFG1-regulated SAP6 gene of C. albicans which encodes for a unique secreted aspartyl
16
proteinase. They concluded that proteases contribute to corneal pathogenicity in C. albicans
17
keratitis and are also associated with morphogenic transformation from yeast to invasive
18
filamentous forms.
19
3.1.3
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Toxigenicity
Various keratitis causing fungi are known to produce mycotoxins; however their exact role
21
in the pathogenesis of keratitis is still unknown. Several toxins produced by Fusarium spp.
22
include nivalenol, T-2 toxin, deoxynivalenol, diacetoxyscirpenol and fusaric acid. Raza et al., 17
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(1994) studied the relationship of toxin production with the severity of disease and concluded
2
that toxin production in vitro did not relate to the clinical presentation of severity of keratitis and
3
the outcome of the treatment. The toxic effects of aflatoxins on the cornea of chicks has been
4
established (Kant et al., 1984). It was demonstrated that when aflatoxin was administered to
5
chicks, haziness of the cornea and separation of corneal lamellae was observed in addition to
6
infiltration by polymorphonuclear leukocytes. Leema et al., (2010) demonstrated that A. flavus
7
isolated from keratitis patients produce significantly more aflatoxin compared to those of the
8
environmental isolates. However the reason behind this phenomenon is unclear and requires
9
further study.
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The ability of fungi to produce various enzymes could also damage tissues, facilitate invasion and eventually influence the severity and outcome of the disease. Zhu et al., (1990)
12
examined the role of fungal proteases in the pathogenesis of mycotic keratitis. It was observed
13
that the clinical isolate of A. flavus, isolated from a patient with severe keratitis, secrete variety
14
of proteases including serine proteases, cysteine proteases, metalloproteases and concluded that
15
fungal collagenases are the mediator of the severe corneal destruction observed in keratitis.
16
When attempted to compare the presence of fungal proteases in vitro and in vivo, the corneal
17
isolates of A. flavus and F. solani predominantly secreted serine proteases and little
18
metalloproteases in vitro. In vivo, however the protease profile shifted to metalloproteases and no
19
serine protease activity was detected (Gopinathan et al., 2001). Matrix metalloproteinases are
20
thought to play a pathological role in the degradation of extracellular matrix components such as
21
basement membrane collagen; same as those found in corneal basement membrane and stroma
22
(Boveland et al., 2010). They have also been shown to be upregulated in ulcerative fungal
23
keratitis in rabbit as well as in the tear film of horses with ulcerative keratitis and hence are
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thought to play a probable role in the pathogenesis of fungal keratitis (Gopinathan et al., 2001;
2
Ollivier et al., 2004). However, the exact role of fungal proteases in mycotic keratitis needs
3
further investigations.
4
3.2 Treatment
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Fungal keratitis is a complex entity with many considerations when it comes to treatment.
6
On the whole, treatment generally entails of chemotherapy consisting of topical and/or systemic
7
drugs, alone or in combination with surgical treatment. However, in developing countries with
8
limited care and economical barriers, mycotic keratitis is of major concern as it can cause visual
9
loss in a demographic population that has limited access to care. Each antifungal has its own
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benefits and limitations and therefore must be selected carefully based on etiological agent and
11
susceptibility testing. There are several classes of antifungals available like polyenes, azoles and
12
fluorinated pyrimidines, each with their own advantages and disadvantages. Polyenes, including
13
natamycin, nystatin and amphotericin B, disrupt fungal cell by binding to the cell wall ergosterol
14
of both filamentous as well as yeast-like fungi. However their tissue penetrating ability is poor
15
and therefore is mostly recommended in cases of superficial corneal infections (Thomas, 2003;
16
Ansari et al., 2013). Administration is every 30 min during the first 24 hours and then every hour
17
for the next 24 hours and then gradually tapered off according to the response. While
18
amphotericin B is commonly administered as a topical solution, intracameral administration is an
19
effective alternative in reducing time to disappearance of hypopyon and final improvement
20
(Yoon et al., 2007). Although active against both forms of fungi, it has shown variable activity
21
against Fusarium species. Owing to its side effect profile and lack of coverage against Fusarium
22
keratitis, it is not considered as a first line agent (Ansari et al., 2013). Natamycin on the other
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hand has a broad-spectrum activity against filamentous fungi and is the first line treatment
2
against fungal keratitis and the drug of choice for Fusarium keratitis. The drug can only be given
3
topically and hence can only treat superficial fungal keratitis as opposed to deep stromal fungal
4
invasion. The presence of deep lesions may therefore require other antifungals like azoles
5
administered via subconjunctival or intravenous routes (Tanure et al., 2000).
The azoles including imidazoles and triazoles, at low concentrations inhibit ergosterol
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biosynthesis while, cause direct damage to cell walls at higher concentrations (Tatsumi et al.,
8
2013). Fluconazole and ketoconazole show good intraocular penetration and are therefore good
9
agents against keratitis with deep lesions (Panda et al., 1996). They are the preferred treatment
10
against both candida and filamentous fungi however, fluconazole has narrow coverage against
11
filamentous organisms but ketoconazole is active against Aspergillus, Candida as well as
12
Curvularia species (Rao et al., 1997; Fitzsimons & Peters, 1986; Ansari et al., 2013).
13
Voriconazole is a good alternative with minimal toxicity and is not only active against Candida
14
but also against filamentous fungi such as Fusarium spp. (Marangon et al., 2004; Lalitha et al.,
15
2007). In refractory FK cases, topical voriconalzole has been used as an adjunct to natamycin
16
along with intrastromal injections of voriconalzole with success (Hariprasad et al., 2008).
17
Hariprasad et al., (2008) reviewed 40 case-reports and concluded that voriconalzole is a safe
18
alternative against major ocular fungal infections but shouldn’t be used as a single agent for
19
initial treatment.
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The fluorinated pyrimidines such as flucytosine etc. are converted into thymidine analog
21
blocking fungal thymidine synthesis. They are usually administered with an azole or
22
amphotericin B for synergistic effects and to avoid development of resistance. Subconjunctival 20
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injections have also been used in patients with severe keratitis. Regardless of the drug choice,
2
successful therapy requires prolonged frequent drug administration and therefore adherence of
3
patients to treatment is also an important factor affecting the outcome of treatment. In addition,
4
liposomal preparation of anti-fungal drugs are also under trail and has demonstrated some
5
efficacy in rabbit models (Habib et al., 2010; Abdel-Rhaman et al., 2012). Furthermore Rasool et
6
al., (2012) compared the efficacy of topical clotrimazole-β-cyclodextrin (CBC), a comparatively
7
new anti-fungal compared to amphotericin B and concluded that CBC reduces the period of
8
treatment on average by a duration of 1 week. The authors also reported greater efficacy of CBC
9
in cases of Candida keratitis.
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Surgical interventions are required in case of complications of acute infectious processes as well as if the disease is refractory to medical management. Penetrating keratoplasty (PK) is the
12
most common surgical treatment used to excise lesions of the cornea and replaced with a donor
13
corneal graft in case of refractory or severe cases of fungal keratitis (Prakash et al., 2008). In
14
addition, it is an effective way to treat corneal perforations as a result of fungal infections (Xie et
15
al., 2007). As an alternative to PK, debridment is also used where causative agent and
16
necrotizing material is removed thereby enhancing the penetration of anti-fungal medications by
17
the removal of epithelium. It is recommended where the necrotizing tissues are hindrance to the
18
healing of corneal ulcers.
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Alternate surgical procedure includes lamellar keratoplasty (LK) in which only diseased
20
layers of the corneal surface are excised thereby leaving the underlying structures of the cornea
21
intact. It is recommended when the fungal invasion is only focal (Price et al., 2005; Reddy et al.,
21
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1
2013). In thisway, the risk of endothelial rejection, the most severe type of corneal graft
2
rejection, is minimized. The challenging nature of fungal keratitis along with less than ideal outcome of current
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treatment regimes, various experimental advances have also been made. Corneal collagen cross-
5
linking (CXL) is a relatively new technique currently been investigated in conjunction with
6
photo-activated riboflavin (PAR) in refractory keratitis cases (Li et al., 2013; Panda et al., 2012).
7
Investigations into the use of nanoparticle technology with terbinafine and silver have also
8
shown promising results (Tayel et al., 2013; Xu et al., 2013). In addition, topical aqueous garlic
9
extracts have been tested on rabbit keratitis models infected with A. flavus (Ismaiel et al., 2012).
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Nonetheless, fungal keratitis is a complex entity with many considerations when it comes to
11
treatment. However, prompt identification of the etiological agent along with targeted therapy
12
may result in favourable outcome.
13
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Bacterial keratitis
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The major bacterial agents of infectious keratitis include Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumonia and Serratia species (Wong et al., 2012).
16
The community acquired cases of bacterial keratitis are usually resolved with an empirical
17
treatment, however if left un-attempted, may result in perforation, endophthalmitis and loss of
18
vision (Callegan et al., 1994; Wong et al., 2012). Clinical presentation of bacterial keratitis
19
include acute pain, redness, photophobia and corneal ulceration (Stapleton et al., 2007).
20
Pseudomonas ulcers are more severe at presentation than other bacterial ulcers and are often
21
difficult to treat, resulting in worse visual outcome than other bacterial ulcers (Sy et al., 2012;
22
Green et al., 2008).
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Bacterial keratitis accounts for approximately 90% of all microbial keratitis cases (Musa et al., 2010) with P. aeruginosa as the most common culprit, worldwide (Dutta et al., 2012).
3
Corneal infection of Pseudomonas is most commonly associated with the use of contact lenses
4
and was rarely reported as a problem prior to the emergence of contact lens (Marquart &
5
Callaghan, 2013; Ramirez et al., 2012). The resistance of Pseudomonas to disinfectants, coupled
6
with its adherence capability to plastics, facilitates its introduction into the eye where it can react
7
with defective corneal epithelium, gaining further entrance into the corneal stroma. S. pneumonia
8
on the other hand is the major cause of corneal ulcers in developing countries however, some
9
reports emphasize on the fact that Streptococcus are most commonly encountered after P.
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aeruginosa and/or S. aureus eye infections (Bharathi et al., 2010; Yilmaz et al., 2007; Marquart
11
& Callaghan, 2013). Unlike P. aeruginosa, pneumococcal keratitis is not commonly associated
12
with the use of contact lenses and the predisposing factors often include ocular trauma or surgery
13
(Marquart and Callaghan, 2013; Wagoner et al., 2007; Cosar et al., 2001; Lifshitz et al., 2005;
14
Mulet et al., 2010; Rehany et al., 2004).
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Along with P. aeruginosa, S. aureus is also a common etiological agent (Marquart & Callaghan, 2013; Liesegang, 1998; Wong et al., 2011; Jeng et al., 2010; Palmer & Hyndiuk,
17
1993). It is a commensal organism that can readily gain access into the eye, given the
18
opportunity (Mohammadinia et al., 2012; Tacconelli & Johnson, 2011; Speaker et al., 1991).
19
Individuals whose eyes are compromised due to various reasons including ocular surgery or
20
trauma, contact lens use, viral infection or other eye illnesses are at high risk of developing this
21
infection (Cheung & Slomovic, 1995; Ormerod et al., 1987; Coster & Badenoch, 1987). In
22
particular, the ability of S. aureus to develop antibiotic resistance makes this infection among the
23
most difficult one to treat (Asbell et al., 2008). This, along with Serratia marcescens, which once
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was considered a harmless saprophyte, has now been increasingly isolated from the corneal
2
surfaces of keratitis patients (Parment, 1997). The isolation rate of S. marcescens from contact
3
lens-coupled corneal infections ranges from 5 – 28%; almost comparable to the isolation rate of
4
P. aeruginosa in contact lens-related keratitis (Schein et al., 1989; Hume et al., 2007). S.
5
marcescens is a motile gram negative rod abundantly dispersed in nature and contributes to
6
contact lens-related keratitis (Parment et al., 1996; Parment, 1997).
7
4.1 Mechanism
8
4.1.1
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Bacteria initiate infection by engaging with the host cell-surface receptors via various
10
adhesins, which mediate bacterial binding to corneal epithelial cells. Microbial adhesins not only
11
play a role in bacterial attachment to the surface of epithelial cells but may also play an active
12
role in subsequent interactions and infective process. They may act as toxins initiating microbial
13
invasion and contribute to subsequent pathogenic cascade. Bacteria display several adhesins on
14
their surface such as pili or fimbriae, which recognize specific carbohydrates or proteins on the
15
surface of host cell. The adherence of P. aeruginosa, S. pneumonia and S. aureus is significantly
16
higher to damaged corneal epithelium compared to other bacteria accounting for their frequent
17
isolation from keratitis cases.
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Studies have shown that purified pili successfully compete with cold bacteria for binding to
19
ocular surface and have been used to protect against P. aeruginosa keratitis. Corneal epithelial
20
glycoproteins act as surface receptor mediating pilus binding activity and amino sugar sialic acid
21
was able to completely inhibit pilus binding to mouse corneal epithelial cells (Hazlett et al.,
24
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1992; Rudner et al., 1992). Bacterial flagella on the other hand are filamentous organelles,
2
responsible for bacterial motility and therefore are also responsible for dissemination of
3
infection. More than 95% of P. aeruginosa clinical isolates are flagellated and flagella-deficient
4
mutants have been observed to be non-virulent (Ansorg et al., 1984). In addition the anti-
5
flagellar antibody homologous to the infecting strain protects mice from Pseudomonas corneal
6
infection (Rudner et al., 1992). Furthermore, the glycocalyx may also play an important role in
7
bacterial adhesion by producing slime aggregates resistant to phagocytosis thereby enabling
8
them to adhere to cells (Hyndiuk, 1981).
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S. aureus surface adhesins, collectively known as MSCRAMMS (microbial surface components recognizing adhesive matrix molecules), have also been recognized to mediate
11
bacterial adherence to host extracellular matrix components, collagen, fibronectin, fibrinogen,
12
laminin and elastin (Jett & Gilmore, 2002). Rhem et al., (2000), studied the role of MSCRAMM
13
Cna (collagen-binding adhesin) in S. aureus keratitis and concluded that collagen-binding
14
adhesin is a virulence factor for S. aureus keratitis and is involved in the early events of
15
pathogenesis of S. aureus infection of the cornea. Similarly, Jett & Gilmore (2002) studied the
16
role of S. aureus fibronectin-binding protein as an epithelial and/or endothelial cell
17
adhesin/invasion protein. Their data suggested that FnBPs is a surface ligand for human corneal
18
cells and play a key role in host-parasite interactions. It serves as an important adhesin and
19
triggers invasion in ulcerative keratitis caused by S. aureus.
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Likewise, Streptococci colonize different sites on human body by expressing multiple
21
adhesins and hence their attachment to human tissues is mediated by a diverse group of bacterial
22
surface proteins, MSCRAMMS (Hammerschmidt, 2006). Plasmin and fibronectin binding 25
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protein A (PfbA), a MSCRAMMS, is known to promote adherence and invasion of bacteria to
2
human epithelial cells by recognizing molecules like fibronectin. In addition, pneumococcal
3
surface adhesin A (PsaA), pneumococcal surface protein A (PspA), pneumolysin (ply),
4
pneumococcal adherence and virulence factor A (PavA), choline-binding protein A
5
(CbpA/PcpA), putative protease maturation protein A (PpmA), IgAI protease (IgAIp), and the
6
streptococcal lipoprotein rotamase A (SIsA) have all been shown to be associated with
7
pneumococcal adherence and virulence (Rajam et al., 2008). Streptococcal cell surface may
8
contain fibrillar structure like pili and fibrils which may also mediate attachment to cell surface
9
to initiate infection. The pneumococcal surface protein C (PspC) promotes adherence and uptake
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of pneumococci into nasopharyngeal epithelial cells (Hammerschmidt et al., 2000). Also,
11
antibodies against PsaA which code for pneumococcal surface adhesin A (PsaA) resulted in
12
reduced adherence to nasopharyngeal epithelial cells (Romero-Steiner et al., 2003). Therefore,
13
these adhesins are likely involved in the initial stages of Streptococcal ocular infection.
14
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Serratia marcescens on the other hand possess mannose-specific adhesins, associated with flagella. These adhesins probably interact with mannose glycoproteins expressed on the surface
16
of corneal epithelial cells as a result of abrasion or trauma due to the use of contact lenses.
17
Labbate et al., (2007), identified type I fimbriae as the critical adhesin that mediate attachment of
18
S. marcescens to human corneal epithelial cell surface. They also identified two AHL (N-
19
acylhomoserine lactone)-regulated genes, bsmA and bsmB, to be involved in adhesion to biotic
20
surface and the expression of these genes leads to the attachment of S. marcescens to HCE cells.
21
In 2007, Shanks et al., demonstrated the role of oxyR and type I fimbrial genes and concluded
22
that they are involved in cell-cell and cell-biotic surface interactions. In addition, two classes of
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pili, mannose-resistant and mannose-sensitive pili may also play a role in the attachment of
2
bacteria to epithelial cells (Hejazi & Falkiner, 1997).
3
4.1.2
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Bacterial invasion and cytotoxic effects Once adhered to the epithelial surface, the pathogen invades into the corneal stroma. This
invasion is facilitated via proteases, exotoxins resulting in degradation of basement membrane
6
and extracellular matrix, causing cells to lyse. A number of exotoxins including heat-stable
7
haemolysin, phospholipases, exotoxins play a role in invasion of bacteria into the cornea with
8
eventual stromal necrosis (O’Brien, 2003). Once bacterial invasion into the cornea has ensued,
9
the infection progresses rapidly towards melting of cornea facilitated by bacterial proteases,
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activation of metalloproteases and stimulation of immune response resulting in further damage
11
via release of reactive oxygen intermediates and host proteases (Thidodeaux et al., 2007;
12
Marquart & Callaghan, 2013). Proteases contribute to the pathogenesis of keratitis by degrading
13
basement membrane, laminin, proteoglycans, collagen, and extracellular matrix (Heck et al.,
14
1986; Twining et al., 1986).
P. aeruginosa is capable of secreting at least 7 different proteases including elastase A and
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B, modified elastase, alkaline protease, protease IV, P. aeruginosa small protease and large
17
exoprotease, some of which play a potential role in the pathogenesis of keratitis (Twining et al.,
18
1993; Marquart et al., 2005). Metalloproteases especially elastase B (Las B) and alkaline
19
protease (AP), play a considerable role in keratitis as Las B injection into the corneal stroma
20
result in significant corneal damage (Ijiri et al., 1993; Kessler et al., 1977). Protease IV (PIV) on
21
the other hand cleaves variety of host defense proteins including immunoglobulins, complement
22
components, antimicrobial peptides and surfactants and hence contributes to the virulence of the
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organism (Engel et al., 1998; Alionte et al., 2001). Pseudomonas aeruginosa small protease
2
(PASP) is able to cleave collagen, the chief structural component of the corneal stroma and
3
hence it could play an important role in the obliteration of cornea (Tang et al., 2009). Purified
4
PASP when injected into the rabbit cornea resulted in the destruction of the epithelium and the
5
formation of erosions that can reach into the stroma. These findings suggest that PIV provides
6
bacteria with a defense arm against multiple host defense molecules, while PASP degrads
7
collagen-based structural component of the eye, thereby mediating the pathophysiology of
8
keratitis.
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Mochizuki et al., (2014) determined the role of MucD protease of P. aeruginosa in
10
keratitis in the cornea of mice. The number of bacteria and clinical score in eyes infected with
11
mucD deficient strain was significantly less compared to the parent or rescued strain. In addition
12
large number of infiltrating PMN cells were observed in mucD deficient eyes along with higher
13
MIP2 levels. It was concluded that MucD protease of P. aeruginosa suppressed IL-1β, KC and
14
MIP2 as well as inhibited neutrophil recruitment in the cornea and hence plays a critical role in
15
P. aeruginosa keratitis by facilitating the evasion of immune response.
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Karthikeyan et al., (2013), examined the expression of three main exotoxins, ExoS, ExoT and ExoU, in clinical isolates of P. aeruginosa and showed that majority of strains (84%) were
18
ExoS+ and ExoT+ but lacked ExoU. However, 5 out of 7 strains that were expressing ExoU
19
were also positive for ExoS which is usually a rare phenotype. These exotoxins are directly
20
injected into the host cell via type III secretion system of P. aeruginosa where they exert their
21
cytotoxic effects onto the host cell (Hauser, 2009). ExoU expressing strains cause rapid cell lysis
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1
whereas, ExoS strains are invasive in nature causing membrane blebbing within epithelial cells
2
which are utilized as a site for bacterial replication and motility (Fleiszig et al., 1996). The capsular polysaccharide of S. pneumoniae which once was considered the central
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dogma in pneumococcal virulence is proven to be untrue in the case of keratitis; as noncapsular
5
strains of S. pneumoniae are also able to cause severe keratitis in rabbit keratitis infection models
6
(Norcross et al., 2010; Reed et al., 2005). Other than capsule, the most studied virulence factor
7
of pneumococcus is pneumolysin, a toxin belonging to the family of bacterial cholesterol-
8
dependent cytolysin. It causes direct cell damage by binding to the cholesterol in the host cell
9
membrane followed by polymerization into 30-50 mers, thereby forming pores in the host cell
10
membranes (Morgan et al., 1995; Tilley et al., 2005). In addition to forming pores, it initiates
11
immune-derived damage by activating complement system and induces inflammation (Paton et
12
al., 1984). Futhermore, reduced corneal virulence was demonstrated by of pneumolysin-deficient
13
strain of S. pneumoniae in a rabbit model of intrastromal infection. It was shown that mutation in
14
the complement activation domain of pneumolysin resulted in reduced polymorphonuclear
15
leukocyte (PMN) recruitment and therefore less corneal damage and virulence compared to the
16
wild-type strain (Johnson et al., 1995).
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Proteins other than pneumolysin, includes choline-binding proteins like pneumococcal
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surface proteins A and C (PspA and PspC), neuraminidase A (NanA) and a variety of other
19
surface-associated proteins such as those involved in adherence, immune evasion, activation and
20
enzymatic reactions have been identified to be involved in non-ocular models of infection
21
Recently a metalloprotease, ZmpC, has been shown to induce ectodomain shedding of the
22
membrane-associated mucin (MAM) from conjunctival and corneal epithelial cells leading to the 29
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loss of glycocalyx barrier function and enhanced internalization of bacterium. It was concluded
2
that the removal of MAMs may serve to be an important virulence mechanism employed by S.
3
pneumoniae (Govindarajan et al., 2012).
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The alpha-toxin is a pore-forming lytic cytotoxin produced by nearly all strains of S. aureus. Once it gains entry into the cytoplasmic membrane, it moves laterally until seven
6
subunits combine in a circular arrangement (Menestrina et al., 2001; Pany et al., 2004;
7
Vijayvargia et al., 2004). The individual toxin molecules interact with cavoline-1, thereby
8
forming pores in the membrane. Callegan et al., (1994) studied the role of alpha-toxin in the
9
corneal virulence of S. aureus and concluded that it plays a major role in staphylococcal keratitis,
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causing destruction of corneal tissues in the infected eye. Later, Moreau et al., (1997) showed
11
that even nanogram quantity of purified alpha-toxin causes extensive sloughing of the rabbit
12
corneal epithelium, corneal edema and severe iritis. Gamma-toxin, a two component toxin from
13
S. aureus is composed of an F component and an S component that are non-toxic on their own.
14
The S component binds to the target cell followed by binding of the F component both of which
15
then move laterally in the cell membrane joining the other F-S pairs to form a ring that penetrates
16
the membrane resulting in cell lysis (Gravet et al., 1998; Szmigieski et al., 1999). The
17
pathogenic role of gamma- and alpha-toxin were determined by Dajcs et al., (2002) who
18
illustrated that the virulence of strain, Newman is mediated by both gamma- and alpha-toxin,
19
with alpha-toxin mediating corneal epithelial erosions. The strain deficient in either toxins were
20
considerably less virulent compared to parent or rescued strains. S. aureus secretes several two
21
component toxins each of which has its own S and F components (Gravet et al., 1998). This two
22
component toxin system of S. aureus is complicated by the fact that S component of one toxin
23
can also bind to the F component of the other toxin thereby creating several unique combinations
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with their own specific toxicity. The role of these toxins in corneal virulence is yet to be
2
determined. The protein produced by setnm-1 gene has been shown to cause extensive corneal
3
damage; an attribute mediated by its protease activity and is now considered to be important in
4
the virulence of S. aureus keratitis (Caballero et al., 2008). In addition, mutant deficient in this
5
gene is considerably less virulent compared to its parent or rescued strain (Caballero et al.,
6
2010).
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Similarly, intra-corneal injections of sub-microgram amounts of Serratia metalloprotease were able to elicit a rapid and extensive corneal tissue damage of rabbit corneas by causing
9
liquefactive necrosis and descemetocele formation (Kreger & Griffin, 1975; Lyerly & Kreger,
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1979). Lyerly et al., (1981) showed that acute inflammation, liquefactive necrosis of rabbit
11
cornea and descemetocele formation occurred after intra-corneal injection of Serratia protease.
12
In addition, they also showed the in vitro solubilization of the stromal proteoglycan ground
13
substance. Ultramicroscopic examination of damaged cornea revealed loss of ruthenium red
14
staining of the proteoglycan ground substance and dispersal of ultra-structurally normal collagen
15
fibrils. Therefore, the major corneal damage after the injection of Serratia protease is due to the
16
solubilization and loss of ground substance of the tissue and the proteases are at least in part
17
involved in the production of severe corneal damage caused by this bacterium. Kamata et al.,
18
(1985) identified a 56-kDa protease as one of the major factors contributing to the pathogenesis
19
and tissue destruction caused by this bacterium. Vaccination of rabbits with purified protease
20
preparations from S. marcescens exhibited significantly less corneal destruction upon corneal
21
encounter with live bacteria, indicating that proteases are important virulence factors during the
22
development of serratial keratitis (Kreger et al., 1986). Marty et al., (2002) showed that mutant
23
strains of S. marcescens that are deficient in the production of 56 kDa metalloprotease are
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considerably less cytotoxic to mammalian cells. It was further revealed that the culture filtrates
2
of wild-type bacteria pre-treated with EDTA or 1,10-phenanthroline (inhibitor of
3
metalloproteases) exhibited significantly reduced cytotoxicity.
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Gram negative lipopolysaccharide is also an important virulence factor in infectious
5
keratitis. It contributes to the pathogenesis of keratitis by mediating the attachment to the cornea
6
and contact lenses, bacterial internalization and survival inside corneal epithelial cells. In
7
addition, it confers resistance to the complement-mediated killing and stimulates neutrophil
8
migration and infiltration into the cornea with subsequent corneal scarring and opacification.
9
Also, exopolysaccharide formation by both gram positive and gram negative bacteria results in
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local immunosuppressive effects as well as interference with phagocytosis.
11
4.1.3
Stromal necrosis and production of ring infiltrate
Bacterial exotoxins and proteases are constitutively released during multiplication of
13
bacteria. These toxins and enzymes persist in the cornea for a prolonged period of time causing
14
continual stromal destruction and can deprive eye of its vision. Most exotoxins are heat-labile
15
and have antigenic properties. The LPS, an endotoxin within the cell wall of gram negative
16
bacteria is released resulting in the production of stromal rings. These rings consist of
17
polymorphonuclear leucocytes within the corneal stroma, which are chemo-attracted by the
18
alternate complement pathway.
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4.2 Treatment
20 21
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Due to the rapid progression of bacterial keratitis, it should be treated as a medical emergency and empirical antibiotic treatment should be promptly commenced. The objective for 32
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the initial therapy is the rapid eradication of the corneal pathogen. Basically, no single antibiotic
2
is effective against all bacterial keratitis-causing organism and therefore an agent having broad
3
spectrum activity covering both, gram negative and gram positive organisms is desirable.
4
Broadly speaking, two treatment options are available; fluoroquinolones monotherapy and/or
5
combination therapy of fortified antibiotics including cefazolin and tobramycin or gentamicin
6
(Gokhale, 2008). The aminoglycosides in fortified drops provides excellent coverage against
7
gram-negative organisms and are also active against Staphylococci and some Streptococci.
8
Cephalosporin on the other hand gives good coverage for non-penicillinase producing gram-
9
positive bacteria. Fluoroquinolone antibiotics have a broad spectrum activity against both gram-
10
negative and gram-positive organisms including penicillinase-producing and methicillin resistant
11
Staphylococci. They inhibit DNA gyrase and topoisomerase IV; the key enzymes involved in
12
DNA replication and transcription leading to bactericidal effect of these antibiotics (Wong et al.,
13
2012; Pestova et al., 2000; Blondeau, 2004).
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The frequency of application of antibiotic drops depends on the severity of infection but usually they are administered every half an hour for the first 24 – 36 hours (O’Brien, 2003;
16
Gokhale, 2008). In severe cases, an initial loading dose is achieved via a drop every 5 min for the
17
first 30 min. The concept behind the loading dose is the vascularity of the cornea and poor
18
penetration of the drug into the corneal stroma which requires frequent topical applications to
19
achieve minimal inhibitory concentration. The therapy is then tapered off gradually based on the
20
clinical response of the patient. Other antibiotics used include amikacin, vancomycin,
21
methicillin, clotrimoxazole, and clarithromycin depending on the causative agent responsible for
22
keratitis (Gokhale, 2008).
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Owing to the rich innervation of the cornea, the disease is frequently associated with
2
considerable pain and therefore analgesics or pain control medications are recommended, which
3
result in improved patient comfort and effective delivery of the treatment regimen. The use of
4
corticosteroids as anti-inflammatory agents are controversial and are best avoided until the
5
infection is completely eradicated or at least under control (O’Brien, 2003; Gokhale, 2008). In
6
patients with corneal ulceration the use of non-steroidal anti-inflammatory drugs should also be
7
avoided due to increased risk of corneal melting and perforation. Topical cycloplegics are used
8
to relieve ciliary spasm, pain and formation of synechiae. Secondary glaucoma may result in
9
keratitis due to increased intraocular inflammation and hence must be treated with topical
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antiglaucoma medications.
In the case of marked corneal ulceration, the temporary use of therapeutic soft contact
12
lenses may facilitate stromal repair and promote re-epithelialization by protecting corneal surface
13
from mechanical trauma. They may act as a tear film antibiotic retention device thereby
14
facilitating penetration by prolonging contact time (Matoba & McCulley, 1985; Busin &
15
Spitznas, 1988). In addition, collagen corneal shields soaked in antibiotic solution have also been
16
used as an effective adjunct and have shown to increase antibiotic penetration (O’Brien et al.,
17
1988; Unterman et al., 1988). Furthermore, temporary intracanalicular collagen implants and
18
liposomal systems have also been designed to prolong drug retention and interaction (Gilbert et
19
al., 1986; Schaeffer & Krohn, 1982).
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Therapy for bacterial keratitis is not only to eradicate the causative agent but also to
21
prevent tissue destruction and irreversible structural alterations caused by enzymes and toxins
22
released by the bacteria. Enzyme inhibitors like disodium edetate, acetylcysteine, Heparin have 34
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been shown to be effective experimentally (Slansky et al., 1971; Ellison & Poirier, 1976; Mehra
2
et al., 1975). Synthetic inhibitors of matrix metalloproteases have been shown to inhibit P.
3
aeruginosa proteases in experimental Pseudomonas keratitis (Barletta et al., 1996). The corneal
4
stroma which constitutes about 90% of corneal thickness is made up of regularly arranged
5
collagen fibrils. Thus the collagenases from bacteria, digest the human collagen causing the
6
cornea to melt. Moreover, collagen cross linking is a useful technique which uses riboflavin and
7
UVA irradiation to strengthen corneal tissues thereby enhancing its rigidity (Spoerl et al., 1998;
8
Spoerl & Seiler, 1999; Wollensak et al., 2003). The interaction between riboflavin and UVA
9
strengthens chemical bond formation between collagen fibrils and hence increasing its resistance
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to enzymatic digestion (Spoerl et al., 2004). In addition, the photoactivation of riboflavin has
11
cidal effect on microorganisms by causing damage to microbial DNA and/or RNA (Wong et al.,
12
2012).
In case of progressive corneal thinning or perforations, less than 2 mm cyanoacrylate tissue
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adhesives may be used, which may also have some inherit antibacterial activity as well
15
(Eiferman & Snyder, 1983; Gokhale, 2008; O’Brien, 2003; Kirkness et al., 1991). Tissue
16
adhesives are only suitable for small perforations, as they are toxic to corneal endothelium. In
17
case of large perforations, therapeutic penetrating keratoplasty may be required. Corneal patch
18
grafting may be an alternative to tissue adhesives and conjunctival flaps may help selected cases
19
of refractory ulceration to assist healing. Severe bacterial keratitis may also result in cataract
20
formation due to bacterial toxins, iridocyclitis and treatment toxicity (Lotti & Dart, 1992).
21
Surgical intervention may be required in such cases depending on the degree of corneal scarring
22
and opacification.
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1
5
Concluding remarks Microbial keratitis, is a complex entity with many considerations when it comes to its
3
treatment. It is a major public health concern particularly in developing countries where access to
4
care is limited and economic barriers are huge where it can become a leading cause of visual loss
5
in a population that is young. As with all corneal infections, proper identification of the causative
6
agent followed by appropriate targeted therapy can abate the risk of complications. A better
7
understanding of the pathogenic cascade would no doubt lead to improved clinical treatment.
8
However the emergence of drug-resistant strains along with the recurrence of infections
9
emphasise on the need for more effective therapeutic modalities. Additionally, the need to
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identify the genetic basis of virulence factors responsible for the disease is also important, as the
11
pathogenicity of keratitis is a sum of multiple events occurring together in time and space for the
12
successful transmission of the pathogen to the susceptible host, overcoming its physiological
13
barriers and eventually producing disease. Subsequently, a complete understanding of
14
pathogenesis of the particular organism will undoubtly lead to the identification of potential
15
therapeutic interventions.
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Acknowledgments: This work was supported by Aga Khan University, Pakistan and Sunway
63
University, Malaysia.
64
Conflict of Interests: The authors declare that there is no conflict of interests regarding the
65
publication of this paper.
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ACCEPTED MANUSCRIPT Highlights Microbial keratitis is caused by bacteria, fungi, and protist pathogens
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Epithelial defects and injuries are key predisposing factors in microbial keratitis
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Factors from the host and the pathogen contribute to the disease.
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Successful prognosis requires early diagnosis and aggressive treatment.
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This is a timely review of our current understanding of microbial keratitis.
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