Hazlett, L. D., Kreindler, F. B., Berk, R. S., and Barrett, R. (1990) Curr. Eye Res. 9:129â138. Hazlett, L. D., Masinick, S., Berk, R. S., and Zheng, Z. (1992a) Exper.
Bioscience Reports, Vol. 21, No. 4, August 2001 ( 2002)
Contact Lens Related Corneal Infections M. D. P. Willcox1,2 and B. A. Holden1 Receiûed April 12, 2001 This article describes microbial keratitis, infection of the cornea by micro-organisms. Contact lens wear is a predisposing factor for the development of microbial keratitis. Microorganisms probably adhere to the contact lens, transfer from the contact lens to a damaged or compromised corneal epithelial surface, penetrate into the deeper layers of the cornea and produce corneal damage. Host responses to the invading micro-organisms, while designed to protect the eye, can often exacerbate the situation and the resulting microbial keratitis can lead to permanent blindness. The microbial, biochemical and immunological aspects of MK will be described in detail. KEY WORDS: Keratitis; Pseudomonas aeruginosa; Staphylococcus aureus; adhesion
INTRODUCTION Contact lenses are a successful form of vision correction. However, under certain circumstances inflammatory adverse responses can occur during lens wear and the most severe of these is contact lens related microbial keratitis (MK). MK has the potential to cause vision loss during contact lens wear. MK has been called many synonyms in the literature, however, a proposal has been developed to standardize these names as microbial keratitis (Holden et al., 2000).
DESCRIPTION OF THE CONDITION There is a wide variety of clinical signs and symptoms associated with contact lens related MK. Some signs and symptoms are common to most cases (Holden et al., 2000). The common symptoms include moderate to severe pain of rapid onset; severe redness of the conjunctiva; blurred or hazy vision; photophobia; discharge; puffy lids. Common signs include corneal infiltration (usually central or paracentral; large irregular infiltrate; diffuse infiltration surrounding epithelial lesion; infiltrate usually anterior to mid-stromal), epithelial loss overlying the infiltrate and cells adhering to the endothelium. MK can also present with distinct clinical features and 1
Co-operative Research Centre for Eye Research and Technology, and Cornea and Contact Lens Research Unit, School of Optometry, University of New South Wales, Sydney, Australia. 2 To whom all correspondence should be addressed. 445 0144-8463兾01兾0800-0445兾0 2002 Plenum Publishing Corporation
Willcox and Holden
Table 1. Some Differential Clinical Features of Contact Lens Related Microbial Keratitis Caused by Different Microorganisms Causative microorganism
P. aeruginosa (and other bacteria)
Large, normally central兾 paracentral, loss of epithelium; satellite lesions
Dense infiltrate underlying epithelial loss; suppurative stromal infiltrate; necrosis; ring infiltrate may be seen
Excessive mucopurulent discharge; corneal oedema; conjunctival chemosis and redness
Loss of epithelium; satellite lesions
Infiltrate with ‘‘cotton wool-like’’ appearance
Oedema of cornea; conjunctival chemosis and redness; endothelial plaques seen
Loss of epithelium; punctuate corneal staining
Dendritic like appearance of infiltrate; patchy stromal infiltrates
Severe pain that may be disproportionate to clinical manifestation; conjunctival chemosis and redness
these may help clinicians identify the suspect pathogens (Table 1). Figure 1 shows a picture of an eye with a contact lens related MK. MAGNITUDE OF THE CONDITION Estimates for the incidence of microbial keratitis depend on the contact lens type being worn. Two major types of contact lenses are commonly worn throughout
Courtesy of L. V. Prasad Eye Institute, Hyderabad, India. Fig. 1. Contact lens related microbial keratitis
Contact Lens Infect.s
the world and these are rigid gas permeable lenses (RGP) and soft hydrogel lenses. These lenses can be worn on either a daily wear schedule (where the wearer removes the lens each night, cleans and disinfects the lenses, stores overnight and refits the same lens the next day), daily disposable wear schedule (the wearer removes the lens at the end of the day, disposes of that lens and inserts a new lens each morning), extended wear schedule (the wearer wears the same lens continuously for, commonly, 6 nights, then removes the lens and inserts a new lens on the seventh day) and continuous wear schedule (wearers wear lenses continuously for 30 nights, then removes the lens and inserts a new lens on the thirty first day). For wearers of RGP on a daily wear (DW) schedule incidence rates of between 0.0007–0.04% per person have been described (Poggio et al., 1989; McRae et al., 1991; Cheng et al., 1999; Nilsson and Montan, 1994). For wearers of RGP on an extended wear (EW) schedule a rate of 0.0024% per person has been reported (MacRae et al., 1991). Soft hydrogel lenses worn on a DW schedule have an incidence rate of between 0.0005 to 0.041% per person (MacRae et al., 1991; Poggio et al., 1989; Nilsson and Montan, 1994; Cheng et al., 1999). Soft lenses worn on an EW schedule have an incidence rate of between 0.042–0.6% per person (Cheng et al., 1999; Nilsson and Montan, 1994; Poggio and Abelson, 1993). The continuous wear and daily disposable wear schedules are recent developments and no reports of incidence rates with these schedules have been published. Another way of analysing differences in risk of wearing different types of contact lenses (RGP vs. hydrogel) and wear schedules (DW vs. EW) is to compare the incidence in studies to that of the lowest incidence. Table 2 gives the incidence rates of contact lens related MK as an increase over incidences reported for contact lens related MK in daily wearers of RGP lenses. EW of hydrogen lenses is associated with the highest incidence of MK. The extended wear of soft hydrogel lenses has an 8.25 increase in MK risk compared to daily wear of soft lenses (Schein et al., 1994). RISK FACTORS FOR CONTACT LENS RELATED MICROBIAL KERATITIS Wong et al. (1997) have written that fungal MK is usually the result of trauma to the eye, whereas bacterial MK is most commonly associated with contact lens Table 2. Relative Incidence of MK in Contact Lens Wearers Compared to Daily Wear of RGP Lenses Study
Mode of wear b
MacRae et al., 1991)
RGP Hydrogel Hydrogel Hydrogel Hydrogel Hydrogel Hydrogel
EW DW EW DW EW DW EW
3.5 0.8 2.7 0.4 3.5 3.2 18.2
(Nilsson and Montan, 1994) (Cheng et al., 1999) a
RGP; rigid gas permeable contact lenses. EW, extended wear; DW, daily wear. c Fold increase, difference in the incidence compared to RGP DW. b
Willcox and Holden
wear. Fungal MK is also more likely to lead to perforation of the globe compared to bacterial MK (Wong et al., 1997), although this is likely due to speed and effectiveness of treatment. Contact lens wear is usually cited as the major risk factor for developing MK in an otherwise healthy eye (Bennett et al., 1998; Dart, 1988; Cruz et al., 1993; McClellan et al., 1989). The incidence of MK associated with contact lenses during 1950–60s was 0% and this had increased to 32% in the 1970s and was up to 52% in the 1980s (Erie et al., 1993). Various factors that occur during lens wear can affect the risk of developing MK. Clearly, from the preceding information, extended wear of hydrogel lenses is a major risk factor. Other risk factors include the use of contaminated lens care solutions and products such as storage cases (Cooper and Constable, 1977; Patrinely et al., 1985; Mondino et al., 1986; Dart, 1988; Mayo et al., 1987; Wilhelmus et al., 1988); Serratia marcescens being particularly resistant to polyquaterium compounds in contact lens solutions (Parment, 1997)); use of corticosteroids during contact lens wear (Chalupa et al., 1987); these would, among other things, decrease the protective function of white blood cells among other things), wearing a therapeutic contact lens in the presence of an epithelial defect (Ormerod and Smith, 1986; this may allow entry of microorganisms—see below), having diabetes (Eichenbaum et al., 1982; presumably due to increased fragility of the corneal epithelium) and smoking (Schein and Poggio, 1990). A hydrogel contact lens induces significant changes in all areas of the cornea (Holden et al., 1985). Lens wear can induce epithelial thinning and production of microcysts in the cornea (Holden et al., 1985). The microcystic response, believed to be dead or dying cells, is associated with wearing lenses that do not allow sufficient oxygen to reach the cornea. Also, wearing hydrogel lenses on an extended wear schedule in an animal model was associated with a significant loss of corneal epithelial cell adhesion as the result of defective epithelial cell basement membrane attachment caused by a decrease in the density of hemidesmosomes (Madigan et al., 1987; Madigan and Holden, 1992). However, the rate of shedding of the epithelial cells appears to be reduced during contact lens wear (Ren et al., 1999a; Ren et al., 1999b; O’Leary et al., 1998). The epithelial cells that are shed during contact lens wear can bind the bacterium Pseudomonas aeruginosa (one of the major causes of microbial keratitis—see below). The number of bacteria binding was dependent on the type of lenses worn, with lenses (either RGP or hydrogel) with low oxygen permeabilities allowing more bacteria to bind to the sloughed epithelial cells (Ren et al., 1999b; Ren et al., 1997). However, this increase in binding was not due simply to an increase in hypoxia, as cells shed under conditions of reduced oxygen without contact lenses did not bind greater numbers of bacteria (Ren et al., 1999b). Thus, other factors, including the mechanical effects of contact lens wear, may be involved in this increase in bacterial adhesion. Further studies examining the effect of hypoxia that occurs during lens wear have demonstrated a direct link with MK. In a rabbit model, a lens contaminated with P. aeruginosa and a closed eye was more effective in inducing corneal infection than corneal wounding, inoculation of bacteria and closing the eye (Solomon et al., 1994). This effect was probably mediated by the increased level of oedema that occurred in the contact lens wearing eye.
Contact Lens Infect.s
TYPES OF MICRO-ORGANISMS ASSOCIATED WITH MK As mentioned earlier, various micro-organisms can cause MK and the clinical manifestation of MK can be related to the broad type of causative agent. Table 3 lists the types of bacteria that have been isolated from MK. This list is extensive and demonstrates that many types of bacteria are able to infect the eye. Protozoa, in particular Acanthamoeba species (Bennett et al., 1998; Cheng et al., 1999; Stapleton et al., 1993) and fungi, in particular Fusarium species, Aspergillus species and Candida species (Wilhelmus et al., 1988) have also been isolated from contact lens related MK but at a much lower frequency than bacteria. Pseudomonas aeruginosa is the most frequently isolated bacterium from contact lens associated MK (Tabbara et al., 2000; Patrinely et al., 1985; Galentine et al., 1984; Schein et al., 1989). However, at least in animal models, the cornea needs to be damaged before the majority of these micro-organisms infect.
SEQUENCE OF EVENTS IN THE DEVELOPMENT OF MICROBIAL (BACTERIAL) KERATITIS The remaining sections of this article will deal with the probable sequence of events that occur during the development of MK, the virulence mechanisms of bacteria that contribute to MK and host defence systems. The most probable sequence of events have been summarized in Fig. 2.
Bacterial Contamination of Contact Lenses The source of the normal and pathogenic microbiota in lens wearers is not always clear, although lens material (Mayo et al., 1987; Sweeney et al., 1999), hands (Hart and Shih, 1987) and ocular contamination have been implicated. The likely route for the normal ocular contamination have been implicated. The likely route for the normal ocular microbiota colonizing contact lenses is via the lid margins, whereas colonization by gram-negative bacteria, including potential agents of microbial keratitis, is likely to be from the domestic water supply (Willcox et al., 1997). An increase in the number of bacteria isolated from the conjunctiva and lids during daily lens wear has been reported (Stapleton et al., 1995; Larkin and Leeming, 1991), although the spectrum of micro-organisms did not differ from non-lens wearing eyes. In a mixed group of lens wearers, an alteration in the types of microorganisms was seen with lens wear (more facultative gram-negative bacteria being isolated) along with an increase in the frequency of negative cultures (Hovding, 1981). This finding has been confirmed for extended wear disposable hydrogel lens users only in a more recent study, where the ocular microbiota was sampled on 7 occasions during 12 months of lens wear (Stapleton et al., 1995). Other studies, however, have reported no differences between wearers and non-lens wearers, although an increase in positive ocular cultures was found in former lens users and in association with certain modes of lens wear and types of disinfection systems (Fleiszig and Efron, 1992).
Willcox and Holden Table 3. Bacteria Isolated from Contact Lens Induced MK Micro-organism
Gram-negative bacteria Acinetobacter calcoaceticus Acinetobacter species Enterobacter aerogenes Escherichia coli Hemophilus influenzae Klebsiella oxytoca Klebsiella pneumoniae Klebsiella species Morganella morgani Moraxella lacunata Moraxella species Proteus mirabilis Proteus morganii Proteus ûulgaris Pseudomonas aeruginosa
Pseudomonas capacia Pseudomonas fluorescens Pseudomonas species
Serratia liquefaciens Serratia marcescens Serratia species Stenotrophomonas maltophilia Gram-positive bacteria Bacillus cereus Bacillus species Coagulase negative staphylococci
Corynebacterium diphtheriae Corynebacterium species (diphtheroids) Micrococcus species Nocardia species Propionibacterium acnes Staphylococcus aureus
Staphylococcus species Streptococcus pneumoniae Streptococcus species Viridans streptococci
Publication (Lemp et al., 1984; Cheng et al., 1999) (Bennett et al., 1998; Ormerod and Smith, 1986) (Cooper and Constable, 1977) (Weissman and Mondino, 1990; Lemp et al., 1984; Cheng et al., 1999; Cooper and Constable, 1977) (Leahey and Jones, 1996; Mondino et al., 1986) (Lemp et al., 1984; Dart, 1988) (Lemp et al., 1984) (Cheng et al., 1999) (Mondino et al., 1986) (Lemp et al., 1984; Simcock et al., 1996; Dart, 1988) (Stapleton et al., 1993) (Lemp et al., 1984) (Alfonso et al., 1986) (Lemp et al., 1984) (Bennett et al., 1998; Lemp et al., 1984; Patrinely et al., 1985; Simcock et al.,1996; Tabbara et al., 2000; Dart, 1988; Nilsson and Montan, 1994, Chalupa et al., 1987, Mondino et al., 1986; Ormerod and Smith, 1986; Cooper and Constable, 1977; Cheng et al., 1999) (Patrinely et al., 1985) (Dunn et al., 1989) (Stapleton et al., 1993; Cohen et al., 1996; Cohen et al., 1987; Donnenfeld et al., 1986; Alfono et al., 1986; Adams et al., 1983) (Cooper and Constable, 1977) (Lemp et al., 1984; Cheng et al., 1999; Dart, 1988) (Ormerod and Smith, 1986) (Cheng et al., 1999; Lemp et al., 1984) (Patrinely et al., 1985) (Ormerod and Smith, 1986; Cheng et al., 1999) (Simcock et al., 1996; Tabbara et al., 2000; Patrinely et al., 1985; Dart, 1988; Maguen et al., 1991; Nilsson and Montan, 1994; Cohen et al., 1987; Mondino et al., 1986; Ormerod and Smith, 1986) (Cheng et al., 1999) (Cohen et al., 1987; Dunn et al., 1989) (Ormerod and Smith, 1986) (Weissman et al., 1984) (Weissman et al., 1984; Dunn et al., 1989; Mondino et al., 1986) (Bennett et al., 1998; Simcock et al., 1996; Tabbara et al., 2000; Patrinely et al., 1985; Nilsson and Montan, 1994; Chalupa et al., 1987; Mondino et al., 1986; Ormerod and Smith, 1986; Cooper and Constable, 1977) (Cheng et al., 1999; Stapleton et al., 1993) (Bennett et al., 1998; Dart, 1998) (Cheng et al., 1999) (Bennett et al., 1998; Dart, 1988; Ormerod and Smith, 1986)
Contact Lens Infect.s
Fig. 2. Sequence of events leading to MK.
The types of particular species of bacteria causing MK has also been investigated. For P. aeruginosa the most common types associated with MK were serotypes 0 :6 (36%) and O :11 (36%) (Mayo et al., 1986). More recently considerable diversity of P. aeruginosa isolates from MK has been demonstrated (Bukanov et al., 1994). Bacterial Adhesion to Contact Lenses The second step in the development of MK is the binding of bacteria to a contact lens. Several studies have examined the ability of bacteria to adhere to contact lenses. Staphylococcus epidermidis or Pseudomonas aeruginosa strains adhere in larger numbers to lenses made from hydroxyethyl methacrylate (HEMA) alone compared to lenses made from HEMA plus methyacrylic acid (Fleiszig et al., 1996a; Cowell et al., 1998b; Taylor et al., 1998) and this may be a function of differing water contents (Cook et al., 1993a, b) or charges of these lens types.
Willcox and Holden
However, a contact lens when inserted into the eye rapidly accumulates proteins, glycoproteins and lipids from the tear film (known as deposits) to its surface. Therefore, it is likely that, other than contamination upon insertion (which is usually by bacteria that are part of the normal microbiota), bacteria adhere to these adsorbed components rather than the contact lens material itself. That is not to say the contact lens material will not still affect adhesion; the types of deposits are likely to be affected by the chemistry of the contact lens. Several investigators have examined whether these deposits affect bacterial adhesion. Total protein does not correlate with adhesion of Pseudomonas aeruginosa to lenses (Mowrey-McKee et al., 1992). However, deposits on lenses did increase adhesion in one study (Butrus and Klotz, 1990), although this may be due to increased surface roughness. No relation between the ability of P. aeruginosa to bind to worn contact lenses and the presence of lysozyme or lactoferrin has been found, although worn lenses did usually increase the adhesion of strains (Williams et al., 1997). Albumin coated onto the surface of contact lenses increased the adhesion of P. aeruginosa (Taylor et al., 1998). Similarly, some strains of Serratia marcescens adhered better to lenses coated in an artificial tear fluid (Hume and Willcox, 1997). Lysozyme adsorbed to a contact lens increases the adhesion of Staphylococcus aureus to lenses (Thakur et al., 1999). In summary, bacteria adhere better to contact lenses made of HEMA only and generally better to worn lenses compared to unworn lenses. Albumin and lysozyme adsorbed to lenses tend to increase adhesion of various types of bacteria that are associated with MK. There is probably also a degree of non-specific adhesion that occurs due to forces such as hydrophobic兾hydrophobic interactions or electrostatic attractive and repulsive forces. Bacterial Adhesion to Corneal Epithelium It is generally accepted that bacteria do not infect an intact corneal surface. There needs to be damage to the epithelium in order for bacteria to initiate infection (Klotz et al., 1989). The bacteria need to transfer from the colonized contact lens to the damaged corneal surface, and adhere to that surface. There have been many studies examining the mechanisms of bacterial adhesion to corneal epithelial cells. Table 4 demonstrates the structures and molecules on both bacterial (Pseudomonas aeruginosa) and corneal epithelial surfaces that have been demonstrated to mediate adhesion. Most research has been performed on P. aeruginosa in this respect. Clearly, there are a variety of ways in which P. aeruginosa can adhere to the corneal epithelium. Pili on P. fluorescens mediate adhesion to corneal epithelium (Reichert et al., 1982). A collagen-binding protein of S. aureus is involved in keratitis (Rhem et al., 2000), presumably through adhesion to either the stroma or epithelium. It would appear that, at least for Pseudomonas aeruginosa, bacteria preferentially bind to the leading edge of the wounded corneas (Spurr-Michaud et al., 1988) and that the polarity of the corneal epithelial cells is critical (Fleiszig et al., 1997a). Further work has demonstrated that protease digestion of corneas can affect the adhesion of P. aeruginosa. Digestion of corneas with trypsin, chymotrypsin, V8 protease, subtilisin A or pronase increases adhesion, whereas proteinase K treatment decreases adhesion (Hazlett et al., 1992a). This would indicate that most proteases
Contact Lens Infect.s
Table 4. P. aeruginosa and Host Molecules Involved in Co-adhesion Bacterial structure兾molecule
Corneal epithelial cell structure兾molecule
Asialo GM1 (mouse not rabbit or human corneas1) Galectin-3
(Gupta et al., 1994)
Lipopolysaccharide Lipopolysaccharide with exposed glucose residue Pili
Pili Pili Flagella Not determined Not determined Not determined a
Not determined Various corneal epithelial proteins (F21, 38, 45, 66, 97, H97 kDa). Glycosylation important Asialo GM1 (mouse not rabbit or human corneas1) 57 kDaa α (2–6) sialylated protein (sialic acid is determinant) Glycosylated proteins, sialic acid is determinant Phosphatidylinositol Phosphatidylserine N-acetyl-mannosamine
(Gupta et al., 1997; Hazlett, 1996) (Zaidi et al., 1996) (Wu et al., 1995)
(Gupta et al., 1994) (Hazlett et al., 1995) (Hazlett and Rudner, 1994) (Panjwani et al., 1996) (Panjwani et al., 1996) (Hazlett et al., 1987)
(Zhao and Panjwani, 1995).
expose receptors on epithelial surfaces that bacteria can adhere to, or disrupt the epithelial surface so that bacteria can bind to underlying surfaces. Treating corneas with elastase or alkaline proteinase from P. aeruginosa also increases adhesion of strains probably due to exposure of lipids as subsequent lipase digestion reduces adhesion (Gupta et al., 1996). Treatment of corneas with the lectin concanavalin A, which binds to α -D-mannose or α -D-glucose residues, also decreases the binding of P. aeruginosa (Blaylock et al., 1990). Thus, bacteria have numerous mechanisms for binding to corneal epithelial cells. However, the corneal epithelial cells need to be treated in some way prior to adhesion. That treatment could be either scratching the surface thereby exposing the underlying lay ers or possibly treatment with proteases (either endogenous to the host or bacterial). Once bound to the outer layers of the corneal, bacteria need to penetrate the deeper layers, including the corneal stroma, and multiply.
Invasion of Bacteria into the Corneal Stroma and Release of Bacterial Toxins and Inflammatory Agents If bacteria fail to enter into the deeper layers of the cornea (i.e., the stroma), they will probably be removed from the corneal surface by the action of the tears and normal epithelial sloughing. Indeed, this appears to be what happens to certain gram negative bacteria, including strains of P. aeruginosa (Willcox and Hume, 1999; Cowell et al., 1998a). Other less virulent strains cause only weak inflammatory events in the cornea, have lower numbers of bacteria, cause decreased endothelial and epithelial necrosis and have a faster restoration of corneal clarity (Hazlett et al., 1985). Again, most work has been performed on P. aeruginosa in relation to its ability to
Willcox and Holden
penetrate into the deeper layers of the cornea. P. aeruginosa can penetrate a wounded mouse cornea within 4 hr of infection (Willcox and Hume, 1999). In order for virulent P. aeruginosa to infect the cornea of mice, between 3B102 to 1B105 bacteria need to be applied to the eye (Preston et al., 1995). Using strains that were genetically modified to be deficient in certain potential virulence factors, lipopolysaccharide, RpoN sigma factor (a protein involved in the translation of DNA genes into messenger RNA) and iron-regulated proteins were important for full virulence in the mouse cornea (Preston et al., 1995). However, strains lacking hemolytic or non-hemolytic phospholipase C were fully virulent indicating that these factors were not important in the disease process (Preston et al., 1995). Presumably the lack of lipopolysaccharide reduced adhesion (and also possibly production of inflammation). The lack of the RpoN sigma factor would have rendered the bacteria unable to translate many genes that are potentially of pathogenic importance including pilus production. Iron is an essential nutrient for bacterial growth, and reducing a bacteria’s response to iron probably resulted in a reduced ability to grow in ûiûo. The first demonstration that P. aeruginosa could enter corneal epithelial cells was reported in 1985 (Stern et al., 1985). This bacterial entry into corneal epithelial cells could be a mechanism for penetrating deeper into the epithelial layers. Certainly, strains can enter corneal epithelial cells and survive for up to 24 hr (Fleiszig et al., 1995). This entry into the corneal epithelial cells has been termed bacterial invasion, although the process seems to be an endocytic process that is dependent on actin microfilaments in the epithelial cells (Fleiszig et al., 1995) and as such may be better described as entry rather than invasion. Clearly both the epithelial and bacterial cells are involved in the process. However, for ease we will continue to describe those strains that enter epithelial cells as invasive. In addition to entry into epithelial cells, certain strains of P. aeruginosa can cause epithelial cell cytotoxicity. The invasive or cytotoxic phenotypes appear to be mutually exclusive (Fleiszig et al., 1996b). More recently, this exclusivity has been shown to be dependent on possession of a number of genes. Invasive strains possess the genes exoS and exoT that encode the ADP-ribosylating toxins ExoS and ExoT (Fleiszig et al., 1997b). Cytotoxic strains only possess the gene exoT (Fleiszig et al., 1997b). Interestingly, more recent work from this laboratory have demonstrated that while invasive strains possess these two genes, the products of these genes prevent invasion of epithelial cells by cytotoxic strains (Cowell et al., 2000). Clearly, there must be strict control on the translation兾transcription of these genes. Both cytotoxicity and invasion involve protein tyrosine kinase activity of epithelial cells (Evans et al., 1998). The cytotoxicity of strains is mediated by a type III secretion system. These systems are common in gram negative bacteria and function when there is intimate contact between bacteria and mammalian cell. Type III secretion systems essentially shoot bacterial molecules, including toxins, straight into the host cell cytosol. Studies have demonstrated that disruption of the type III system prevents cytotoxicity occurring (Hauser et al., 1998). Another protein that probably causes cytotoxicity is Exotoxin A and mutants lacking this protein produce less corneal damage (Ohman et al., 1980).
Contact Lens Infect.s
For S. aureus, it is likely that α toxin (α haeolysin) allows the bacteria to enter the deeper layers of the cornea as this toxin can cause corneal epithelial cell necrosis and apoptosis (Moreau et al., 1997). In addition, this toxin also induces corneal erosions in an animal model (O’Callaghan et al., 1997). Proteases produced by bacteria probably mediate both penetration through the corneal epithelial cells and also allow degradation of the corneal stroma. P. aeruginosa produces metalloproteinases and inhibitors of metalloproteinases supplied exogenously during infection reduce corneal damage in an animal model of keratitis (Barletta et al., 1996). Endogenous corneal matrix metaloproteinase-2 (MMP) is activated by P. aeruginosa proteases (Matsumoto et al., 1992; Matsumoto et al., 1993). P. aeruginose elastase activates both MMP-2 and MMP-9 (Twining et al., 1993) and breaks down corneal proteoglycans and, to a lesser extent, collagen (Kessler et al., 1977). During keratitis in a mouse model, alkaline protease has been observed (Kernacki et al., 1995). O’Callaghan et al. (O’Callaghan et al., 1996) have demonstrated that protease IV from P. aeruginosa is important in the production of keratitis. Mice immunized as the result of either prior corneal infection or immunized with bacterial proteases demonstrate reduced levels of bacterial alkaline protease in corneas (Kernacki et al., 1997). In addition, there were reduced levels of latent MMP-2 and latent or active MMP-9 and, as the result of these suppressions, less corneal destruction (Kernacki et al., 1997). Therefore, bacteria penetrate into the deeper layers of the cornea and cause corneal damage by a variety of mechanisms. The most destructive of which are probably the direct toxicity of bacterial proteins to corneal epithelial cells (and probably stromal fibroblasts) and digestion of the corneal layers by bacterial proteases either directly or as the result of activation of endogenous host proteases. Inflammatory responses are probably activated by a combination of bacterial products such as lipopolysaccharide and the degradation of host proteins which generates chemotactic molecules. Host Response to Infection Infection of the cornea is a two way process with the bacteria causing damage and the host attempting to control infection. However, the host response can be subverted by the infecting pathogen, for example (as described aboûe) by activating latent proteases, or can cause direct destruction or tissue remodeling. There have been a series of excellent studies by groups led by Linda Hazlett and Richard Berk from Wayne State University, Detroit, USA, who have studied the basis for the differences in the ability of mice to restore clarity to corneas after infection with P. aeruginosa (young or BALB兾c mice) or loose sight (old or C57BL兾 6 mice) based on age or genetic make-up. These differences can be mediated by a combination of immunological and biochemical variables. Th1 (e.g. C57BL兾6) responder mice have a severer pathology during infection which progresses to perforation of the globe within seven days compared to Th2 (BALB兾c) responder mice (Hazlett et al., 2000). The Th1 mice have increased numbers of bacteria, CD4C and CD8C T-lymphocytes and polymorphonuclear leukocytes (PMNs) in their corneas. Antibodies to CD4C T-lymphocytes
Willcox and Holden
resulted in a lowering of the clinical score (Kwon and Hazlett, 1997). Macrophages are also reduced in susceptible mice (Hazlett et al., 1992b). Indeed, for white blood cells what appears to be critical is the speed of recruitment, peak numbers and duration of the cellular infiltrate (Hazlett et al., 1992b). In contact lens wearing rabbit eyes the numbers of PMNs recruited to the corneal surface is reduced compared to non-lens wearing eyes (Lawin-Brussel et al., 1995). This may be due to a reduced ability of PMNs migrating from the eye lids to reach the corneal surface (Sloop et al., 1999). In humans, we have shown similar findings (Stapleton et al., 1997; Thakur and Willcox, 2000). Dendritic cells, known as Langerhans cells in the cornea, are not normally present except at the outer limbus area. However, the migration of Langerhans cells into the cornea may contribute to susceptibility (Hazlett et al., 1986). It is interesting to note that several recent publications have demonstrated that wearing contact lenses (at least in two animal models; rabbits or guinea pigs) increases the recruitment of Langerhans cells into the cornea (Hazlett et al., 1996b; Sankaridurg et al., 2000). An inhibitor of MMPs, tissue inhibitor of metaloproteinase-1 (TIMP-1), was increased in the corneas of resistant mice during infection and antibody to TIMP-1 resulted in resistant mice progressing to corneal perforation (Kernacki et al., 1999). Aged mice have a defective alternative complement pathway (Hazlett et al., 1999a), reduced PMN recruitment and microbicidal activity of their PMNs (Hazlett et al., 1990), decreased expression of intercellular adhesion molecule-1 (ICAM-1), an adhesion molecule involved in PMN recruitment (Hobden et al., 1995), and decreased levels of interleukin-1β and interferon-γ production (Hobden et al., 1997). IL-6 also appears to play an important role during MK (Cole et al., 1999a) and TNFα and KC play roles later in the disease process (Cole et al., 2000; Cole et al., 1999b). IL-1β , a pro-inflammatory cytokine, and MIP-2, a chemokine for PMNs, have been shown to be important in protection of the cornea and its destruction during keratitis (Kernacki et al., 2000; Rudner et al., 2000)). Susceptible mice have decreased levels of IgG and IgM in sera and IgG in tears that is specific to P. aeruginosa (Preston et al., 1992). IgA can inhibit the binding of P. aeruginosa to the corneal epithelium (Masinick et al., 1997). The precise epitope that these antibodies were raised to was unknown, although other experiments have demonstrated that antibodies to outer membrane proteins of P. aeruginosa, alkaline protease, elastase or Exotoxin A can reduce the clinical severity of the MK (Moon et al., 1988; Steuhl et al., 1987). Mucin can reduce the adhesion of bacteria to the cornea (Fleiszig et al., 1994). However, once mucin is bound to a contact lens it appears to lose its protective function. Lenses with bound mucin cause a more severe MK response probably due to increases in the numbers of bacteria on the contact lens surface (DiGaetano et al., 1986). Thus, the host response is critical in protecting the cornea, but can also produce some of the pathology associated with MK. Effective protection of the cornea is only afforded using prompt and aggressive treatment with antibiotics. THERAPIES FOR MK Therapy for microbial keratitis is critical and should be initiated as soon as MK is suspected. Antibiotics of choice are broad spectrum as it is often impossible to
Contact Lens Infect.s
culture micro-organisms from the MK lesions. However, several studies have examined the susceptibility of microbial isolates form MK to antibiotics. As early as 1986, it was demonstrated that most strains of P. aeruginosa isolated from MK were resistant to ampicillin, cephalothin, neomycin, tetracycline (Mayo et al., 1986). While most S. epidermidis strains are still susceptible to ciprofloxacin, vancomycin and teicoplanin, most are resistant to penicillin (Sechi et al., 1999). These studies highlight the increasing trend of bacteria to become resistant to antibiotics and there is clearly a need for new antibiotic therapies. Interestingly, lysostaphin has recently been used successfully to treat MK due to S. aureus in a rabbit model (Dajcs et al., 2000). Another interesting line of research has been to examine tears for potential antimicrobials. The rationale being that boosting the eyes own defences with or without combination antibiotic therapy might also prove valuable in treating MK. Tear antimicrobial substances do not work alone, for example the combination of lysozyme and lactoferrin appears to be critical in killing staphylococci (Leitch and Willcox, 1998, 1999). Secretory phospholipase A2 has been shown to be produced by the lacrimal gland (Aho et al., 1996) and to be the principal bactericide for staphylococci in tears (Qu and Lehrer, 1998). CONCLUSION In conclusion, MK is a severe but rare disease associated with contact lens wear. Although rare, it is potentially blinding. Specific mechanisms, either bacterial or host, are now known to contribute to the diseases. Prompt and aggressive therapy is critical to a successful visual outcome. Although bacteria are becoming increasingly resistant to commonly prescribed antibiotics, new treatments are being developed and the basic knowledge gained into pathogenic mechanisms involved in MK can now be utilized to devise new therapies that can lead to improved visual outcome. REFERENCES Adams, C. P., Jr., Cohen, E. J., Laibson, P. R., Galentine, P., and Arentsen, J. J. (1983) Am. J. Ophthalmol. 96:705–709. Aho, H. J., Saari, K. M., Kallajoki, M., and Navalainen, T. J. (1996) Inûestig. Ophthalmol. and Vis. Sci. 37:1826–1832. Alfonso, A. L., Mandelbaum, S., Fox, M. J., and Forster, R. K. (1986) Am. J. Ophthalmol. 101:429– 433. Barletta, J. P. et al. (1996) Inûestig. Ophthalmol. and Vis. Sci. 37:20–28. Bennett, H. G., Hay, J., Kirkness, C. M., Seal, D. V., and Devonshire, P. (1998) Br. J. Ophthalmol. 82:137–147. Blaylock, W. K., Yue, B. Y., and Robin, J. B. (1990) CLAO J. 16:223–227. Bukanov, N., Ravi, V. N., Miller, D., Srivastava, K., and Berg, D. E. (1994) Curr. Eye Res. 13:783–790. Butrus, S. I. and Klotz, S. A. (1990) Curr. Eye Res. 9:717–724. Chalupa, E., Swarbrick, H. A., Holden, B. A., and Sjostrand, J. (1987) Ophthalmology 94:17–22. Cheng, K. H. et al. (1999) Lancet 354:181–185. Cohen, E. J., Fulton, J. C., Hoffman, C. J., Rapuano, C. J., and Laibson, P. R. (1996) Cornea 15:566– 570. Cohen, E. J., Laibson, P. R., Arentsen, J. J., and Clemons, C. S. (1987) Ophthalmol. 94:109–114. Cole, N., Bao, S., Thakur, A., Willcox, M., and Husband, A. J. (2000) Immunol. and Cell Biol. 78:1–4.
Willcox and Holden
Cole, N., Bao, S., Willcox, M., and Husband, A. J. (1999a) Infect. and Immun. 67:2497–2502. Cole, N., Bao, S., Willcox, M., and Husband, A. J. (1992b) Immunol. and Cell Biol. 77:164–166. Cook, A. D., Sagers, R. D., and Pitt, W. G. (1993a) J. Biomed. Mater. Res. 27:119–126. Cook, A. D., Sagers, R. D., and Pitt, W. G. (1993b) J. Biomat. Applic. 8:72–89. Cooper, R. L. and Constable, I. J. (1997) Br. J. Ophthalmol. 61:250–254. Cowell, B. A., Chen, D. Y., Frank, D. W., Vallis, A. J., and Fleiszig, S. M. (2000) Infect. and Immun. 68:403–406. Cowell, B. A., Willcox, M. D., Hobden, J. A., Schneider, R. P., Tout, S., and Hazlett, L. D. (1998a) Exp. Eye Res. 67:347–356. Cowell, B. A., Willcox, M. D., and Schneider, R. P. (1998b) J. of Appl. Microbiol. 84:950–958. Cruz, O. A., Sabir, S. M., Capo, H., and Alfonso, E. C. (1993) Ophthalmol. 100:192–196. Dajcs, J. J., Hume, E. B., Moreau, J. M., Caballero, A. R., Cannon, B. M., and O’Callaghan, R. J. (2000) Inûestig. Ophthalmol. and Vis. Sci. 41:1432–1437. Dart, J. K. (1988) Br. J. Ophthalmol. 72:926–930. DiGaetano, M., Stern, G. A., and Zam, Z. S. (1986) Cornea 5:155–158. Donnenfeld, E. D., Cohen, E. J., Arentsen, J. J., Genvert, G. I., and Laibson, P. R. (1986) CLAO J. 12:1459–49. Dunn, J. P., Jr., Mondino, B. J., Weissman, B. A., Donzis, P. B., and Kikkawa, D. O. (1989) Am. J. Ophthalmol. 108:113–117. Eichenbaum, J. W., Felstein, M., and Podos, S. M. (1982) Br. J. Ophthalmol. 66:663–666. Erie, J. C., Nevitt, M. P., Hodge, D. O., and Ballard, D. J. (1993) Archiûe of Ophthalmol. 111:1665– 1671. Evans, D. J., Frank, D. W., Finck-Barbancon, V., Wu, C., and Fleiszig, S. M. (1988) Infect. and Immun. 66:1453–1459. Fleiszig, S. M. and Efron, N. (1992) Optom. and Vis. Sci. 69:354–357. Fleiszig, S. M., Evans, D. J., Do, N., Vallas, V., Shin, S., and Mostov, K. E. (1997a) Infect. and Immun. 65:2861–2867. Fleiszig, S. M. et al. (1996a) Optom. and Vis. Sci. 73:590–594. Fleiszig, S. M. et al. (1997b) Infect. and Immun. 65:579–586. Fleiszig, S. M., Zaidi, T. S., and Pier, G. B. (1994) Adûan. Exper. Med. and Biol. 350:359–362. Fleiszig, S. M., Zaidi, T. S., and Pier, G. B. (1995) Infect. and Immun. 63:4072–4077. Fleiszig, S. M., Zaidi, T. S., Preston, M. J., Grout, M., Evans, D. J., and Pier, G. B. (1996b) Infect. and Immun. 64:2288–2894. Galentine, P. G., Cohen, E. J., Laibson, P. R., Adams, C. P., Michaud, R., and Arentsen, J. J. (1984) Archiû. Ophthalmol. 102:891–894. Gupta, S. K., Berk, R. S., Masinick, S., and Hazlett, L. D. (1994) Infect. and Immun. 62:4572–4579. Gupta, S. K., Masinick, S., Garrett, M., and Hazlett, L. D. (1997) Infect. and Immun. 65:2747–2753. Gupta, S. K., Masinick, S. A., Hobden, J. A., Berk, R. S., and Hazlett, L. D. (1996) Exp. Eye Res. 62:641–650. Hart, D. E. and Shih, K. L. (1987) Am. J. Optom. and Physiolog. Optics 64;739–748. Hauser, A. R., Fleiszig, S., Kang, P. J., Mostov, K., and Engel, J. N. (1998) Infect. and Immun. 66:1413– 1420. Hazlett, L., Rudner, X., Masinick, S., Ireland, M., and Gupta, S. (1995) Inûestig. Ophthalmol. and Vis. Sci. 36:634–643. Hazlett, L. D. (1996) Trends in Microbiol. 4:418. Hazlett, L. D., Kreindler, F. B., Berk, R. S., and Barrett, R. (1990) Curr. Eye Res. 9:129–138. Hazlett, L. D., Masinick, S., Berk, R. S., and Zheng, Z. (1992a) Exper. Eye Res. 55:579–587. Hazlett, L. D., Masinick-McClellan, S. A., and Barrett, R. P. (1990a) Curr. Eye Res. 19:26–32. Hazlett, L. D., McClellan, S., Kwon, B., and Barrett, R. (2000) Inûestig. Ophthalmol. and Vis. Sci. 41:805– 810. Hazlett, L. D., McClellan, S. M., Hume, E. B., Dajcs, J. J., O’Callaghan, R. J., and Willcox, M. D. (1999b) Exper. Eye Res. 69: 575–577. Hazlett, L. D., Moon, M. M., Dawisha, S., and Berk, R. S. (1986) Curr. Eye Res 5:343–355. Hazlett, L. D., Moon, M. M., Strejc, M., and Berk, R. S. (1987) Inûestigat. Ophthalmol. and Vis. Sci 28:1978–1985.
Contact Lens Infect.s
Hazlett, L. D. and Rudner, X. L. (1994) Ophthalmic Res. 26:375–379. Hazlett, L. D., Zelt, R., Cramer, C., and Berk, R. S. (1985) Ophthalmic Res. 17:289–296. Hazlett, L. D., Zucker, M., and Berk, R. S. (1992b) Ophthalmic Res. 24:32–39. Hobden, J. A., Masinick, S. A., Barrett, R. P., and Hazlett, L. D. (1995) Inûestig. Ophthalmol. and Vis. Sci. 36:1107–1114. Hobden, J. A., Masinick, S. A., Barrett, R. P., and Hazlett, L. D. (1997) Infect. and Immun. 65:2754– 2758. Holden, B. A., Sankaridurg, P., and Jalbert, I. (2000) In: Silicone Hydrogels: The Rebirth of Continuous Wear Contact Lenses (D. F. Sweeney, ed.) Butterworth Heinemann, Oxford, pp. 150–213. Holden, B. A., Sweeney, D. F., Vannas, A., Nilsson, K. T., and Efron, N. (1985) Inûestigat. Ophthalmology and Vis. Sci 26:1489–1501. Hovding, G. (1981) Acta Ophthalmologica 59:387–401. Hume, E. B. and Willcox, M. D. (1997) Austral. and N. Zeal. J. Ophthalmol. 25:S39–S41. Kernacki, K. A., Barrett, R., and Hazlett, L. D. (1999) Inûestig. Ophthalmol. and Vis. Sci. 40:3168–3176. Kernacki, K. A., Barrett, R. P., Hobden, J. A., and Hazlett, L. D. (2000) J. Immunol. 164, 1037–1045. Kernacki, K. A., Fridman, R., Hazlett, L. D., Lande, M. A., and Berk, R. S. (1997) Curr. Eye Res. 16:289–297. Kernacki, K. A., Hobden, J. A., Hazlett, L. D., Fridman, R., and Berk, R. S. (1995) Inûestig.. Ophthalmol. and Vis. Sci. 36:1371–1378. Kessler, E., Kennah, H. E., and Brown, S. I. (1977) Inûestig. Ophthalmol. and Vis. Sci. 16:488–497. Klotz, S. A., Au, Y. K., and Misra, R. P. (1989) Inûestig. Ophthalmol. and Vis. Sci. 30:1069–1074. Kwon, B. and Hazlett, L. D. (1997) J. Immunol. 159:6283–6290. Larkin, D. F. and Leeming, J. P. (1991) Eye 5:70–74. Lawin-Brussel, C. A., Refojo, M. F., Leong, F. L., and Kenyon, K. R. (1995) Cornea 14:355–359. Leahey, A. B. and Jones, D. (1996) CLAO J. 22:213–214. Leitch, E. C. and Willcox, M. D. (1988) J. Med. Microbiol. 47:837–842. Leitch, E. C. and Willcox, M. D. (1999) J. Med. Microbiol. 48:867–871. Lemp, M. A., Blackman, H. J., Wilson, L. A., and Leveille, A. S. (1984) Ophthalmol. 91:60–63. MacRae, S. et al. (1991) Am. J. Ophthalmol. 111:457–165. Madigan, M. C. and Holden, B. A. (1992) Inûestig. Ophthalmol. and Vis. Sci. 33:314–323. Madigan, M. C., Holden, B. A., and Kwok, L. S. (1987) Curr. Eye Res. 6, 1257–1260. Maguen, E. et al. (1991) Ophthalmol. 98:1685–1689. Masinick, S. A., Montgomery, C. P., Montgomery, P. C., and Hazlett, L. D. (1997) Inûestig. Ophthalmol. and Vis. Sci. 38:910–918. Matsumoto, K., Shams, N. B., Hanninen, L. A., and Kenyon, K. R. (1992) Curr. Eye Res. 11:1105– 1109. Matsumoto, K., Shams, N. B., Hanninen, L. A., and Kenyon, K. R. (1993) Inûestig. Ophthalmol. and Vis. Sci. 34:1945–1953. Mayo, M. S., Cook, W. L., Schlitzer, R. L., Ward, M. A., Wilson, L. A., and Ahearn, D. G. (1986) J. Clin. Microbiol. 24:372–376. Mayo, M. S., Schlitzer, R. L., Ward, M. A., Wilson, L. A., and Ahearn, D. G. (1987) J. Clin. Microbiol. 25:1398–1400. McClellan, K. A., Bernard, P. J., and Billson, F. A. (1989) Austral. and N. Zeal. J. Ophthalmol. 17:413– 416. Mondino, B. J., Weissman, B. A., Farb, M. D., and Pettit, T. H. (1986) Am. J. Ophthalmol. 102:58–65. Moon, M. M., Hazlett, L. D., Hancock, R. E., Berk, R. S., and Barrett, R. (1988) Inûestig. Ophthalmol. and Vis. Sci. 29:1277–1284. Moreau, J. M., Sloop, G. D., Engel, L. S., Hill, J. M., and O’Callaghan, R. J. (1997) Curr. Eye Res. 16:1221–1228. Mowrey-McKee, M. F. et al. (1992) CLAO J. 18:87–91. Nilsson, S. E. and Montan, P. G. (1994) CLAO J. 20:97–101. O’Callaghan, R. J. et al. (1997) Infect. and Immun. 65:1571–1578. O’Callaghan, R. J., Engel, L. S., Hobden, J. A., Callegan, M. C., Green, L. C., and Hill, J. M. (1996) Inûestig.. Ophthalmol. and Vis. Sci. 37:534–543. Ohman, D. E., Sadoff, J. C., and Iglewski, B. H. (1980) Infect. and Immun. 28:899–908.
Willcox and Holden
O’Leary, D. J., Madgewick, R., Wallace, J., and Ang, J. (1998) Optometry and Vis. Sci. 75:692–696. Omerod, L. D. and Smith, R. E. (1986) Archiû. Ophthalmol. 104:79–83. Panjwani, N., Zhao, Z., Raizman, M. B., and Jungalwala, F. (1996) Infect. and Immun. 64: 1819–1825. Parment, P. A. (1997) Acta Ophthalmologica Scandinaûica 75:67–71. Patrinely, J. R., Wilhelmus, K. R., Rubin, J. M., and Key, J. E. d. (1985) CLAO J. 11:234–236. Poggio, E. C. and Abelson, M. (1993) CLAO J. 19:31–39. Poggio, E. C. et al. (1989) N. Engl. J. Med. 321:779–783. Preston, M. J. et al. (1995) Infect. and Immun. 63:497–501. Preston, M. J., Kernacki, K. A., Berk, J. M., Hazlett, L. D. and Berk, R. S. (1992) Infect. and Immun. 60:885–891. Qu, X. D. and Lehrer, R. I. (1998) Infect. and Immun. 66:2791–2797. Reichert, R. W., Das, N. D., and Zam, Z. S. (1982) Curr. Eye Res. 2:289–293. Ren, D. H., Petroll, W. M., Jester, J. V., Ho-Fan, J., and Cavanagh, H. D. (1990a) CLAO J. 25:80–100. Ren, D. H., Petroll, W. M., Jester, J. V., Ho-Fan, J., and Cavanagh, H. D. (1999b) CLAO J. 25:73–79. Ren, H. et al. (1997) CLAO J. 23:63–68. Rhem, M. N. et al. (2000) Infect. and Immun. 68:3776–3379. Rudner, X. L., Kernacki, K. A., Barrett, R. P. and Hazlett, L. D. (2000) J. Immunol. 164:6576–6582. Sankaridurg, P. R., Rao, G. N., Rao, H. N., Sweeney, D. F., and Holden, B. A. (2000) Cornea 19:374– 377. Schein, O. D., Buehler, P. O., Stamler, J. F., Verdier, D. D., and Katz, J. (1994) Archiû. Ophthalmol. 112:186–190. Schein, O. D. et al. (1989) Cornea 8:281–285. Schein, O. D. and Poggio, E. C. (1990) Cornea 9:S55–S58; discussion S62–S63. Sechi, L. A., Pinna, A., Pusceddu, C., Fadda, G., Carta, F., and Zanetti, S. (1999) J. Clin. Microbiol. 37:3031–3033. Simcock, P. R., Butcher, J. M., Armstrong, M., Lloyd, I. C., and Tullo, A. B. (1996) Acta Ophthalmologica Scandinaûica 74:183–186. Sloop, G. D., Moreau, J. M., Conerly, L. L., Dajcs, J. J., and O’Callaghan, R. J. (1999) Inûestig. Ophthalmol. and Vis. Sci. 40:385–391. Solomon, O. D. et al. (1994) CLAO J. 20:109–113. Spurr-Michaud, S. J., Barza, M. and Gipson, I. K. (1988) Inûestig. Ophthalmol. and Vis. Sci. 29:379– 386. Stapleton, F., Dart, J. K., and Minassian, D. (1993) CLAO J. 19:204–210. Stapleton, F., Willcox, M. D., Fleming, C. M., Hickson, S., Sweeney, D. F., and Holden, B. A. (1995) Infect. and Immun. 63:4501–4505. Stapleton, F., Willcox, M. D., Sansey, N., and Holden, B. A. (1997) Austral. and N. Zea. J. Ophthalmol. 25:S33–S35. Stern, G. A., Lubniewski, A., and Allen, C. (1985) Archiû. of Ophthalmol. 103;1221–1225. Steuhl, K. P., Doring, G., Henni, A., Thiel, H. J., and Botzenhart, K. (1987) Inûestig. Ophthalmol. and Vis. Sci. 28:1559–1568. Sweeney, D. F. et al. (1999) CLAO J. 25:167–175. Tabbara, K. F., El-Sheikh, H. F., and Aabed, B. (2000) Br. J. Ophthalmol. 84:327–328. Taylor, R. L., Willcox, M. D., Williams, T. J., and Verran, J. (1998) Optomet. and Vis. Sci. 75:23–29. Thakur, A., Chauhan, A., and Willcox, M. D. (1999) Austral. and N. Zeal. J. Ophthalmol. 27:224–247. Thakur, A. and Willcox, M. D. (2000) Exp. Eye Res. 70:255–259. Twining, S. S., Kirschner, S. E., Mahnke, L. A., and Frank, D. W. (1993) Inûestig. Ophthalmol. and Vis. Sci. 34:2699–2712. Weissman, B. A. and Mondino, B. J. (1990) Cornea 9:S25–27; discussion S31–S32. Weissman, B. A., Mondino, B. J., Pettit, T. H., and Hofbauer, J. D. (1984) Am. J. Ophthalmol. 97:476– 481. Wilhelmus, K. R., Robinson, N. M., Font, R. A., Hamill, M. B., and Jones, D. B. (1988) Am. J. Ophthalmol. 106:708–714. Willcox, M. D. and Hume, E. B. (1999) Austral. and N. Zeal. J. Ophthalmol. 27:231–233.
Contact Lens Infect.s
Willcox, M. D., Power, K. N., Stapleton, F., Leitch, C., Harmis, N. and Sweeney, D. F. (1997) Optomet. and Vis. Sci. 74:1030–1038. Williams, T. J., Willcox, M. D., and Schneider, R. P. (1997) Austral. and N. Zeal. J. Ophthalmol. 25:S30– S32. Wong, T. Y., Ng, T. P., Fong, K. S., and Tan, D. T. (1997) CLAO J. 23:275–281. Wu, X., Gupta, S. K., and Hazlett, L. D. (1995) Curr. Eye Res. 14:969–977. Zaidi, T. S., Fleiszig, S. M., Preston, M. J., Goldberg, J. B., and Pier, G. B. (1996) Inûestig. Ophthalmol. and Vis. Sci. 37:976–986. Zhao, Z. and Panjwani, N. (1995) Infect. and Immun. 63:353–355.