COMPARISON OF
TOPICAL GATIFLOXACIN 0.3% EYE DROPS & CIPROFLOXACIN 0.3% EYE DROPS
FOR THE TREATMENT OF BACTERIAL CORNEAL ULCER
(Ophthalmology)
Hypothesis
Topical gatifloxacin 0.3% eye drops is more effective than ciprofloxacin 0.3% eye drops in the treatment of bacterial corneal ulcer.
Aims & Objective
Objectives:
A.AGeneral:
ü To compare the efficacy of topical gatifloxacin 0.3% eye drops and
Ciprofloxacin 0.3% eye drops in the treatment of bacterial corneal ulcer.
B. Specific:
ü To analyze the symptom and sign in both groups.
ü To determine the ulcer healing rate both groups.
ü To asses the clinical improvement in both gatifloxacin and ciprofloxacin group.
Introduction
Cornea is the outermost coat of the eyeball, which is the most vital part for vision. It has tremendous optical importance in the visual function. It is the main part of refractive media that contributes about 74% of total diopteric power of normal human eye (John E. Stuphin et al., 2007). So the corneal health and disease are not less important than that of any vital organ of the body. The cornea has some anatomical add physiological specialties with which it can function without any interruption throughout life. In spite of these specialties the cornea frequently becomes diseased and corneal ulceration is one of the top of the list of corneal disease. So we should give great importance when it becomes diseased.
The avascular, clear anatomic structure of the cornea, with its specialized microenvironment predispose to potential alteration and destruction by invading microorganism by virulence factor and host response factors (C. Stephen Foster, 2005).Bacterial Corneal ulcer is a common sight-threatening condition. A wide variety of bacterial species can cause microbial corneal ulcer. The common organisms are Streptococcus pneumonae, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumonae, Pseudomonas aeruginosa and enterobactereriace. Uncommon organisms are N. gonorrhoae, N. meningitides, Moraxella species, Haemophilus species, Mycobacteriam spp. & Corynebacteriam spp. (C. Stephen Foster, 2005).
Bacterial keratitis has the potential to progress rapidly to corneal perforation. Even small axial lesion can cause surface irregularity & scar that can lead to significant loss of vision. (Jack J. Kanski, 2007). The objective of therapy in bacterial corneal ulcer is rapidly to eliminate the infective organism, reduce the inflammatory response, prevent structural damage to the cornea and promote healing of the epithelial surface. (Jones DB.1979). A large number of active antimicrobial drugs available for the treatment of bacterial corneal ulcer a greater choice for cure with less drug related toxicity while providing alternative choices despite the continuing emergence of drug resistant pathogenic organisms (C. Stephen Foster, 2005).
Different antimicrobial agent used in the treatment of bacterial corneal ulcer are penicillin’s, cephalosporin’s, other ?-lactum antibiotics, glycopeptides, aminoglycosides, macrolides, tetracycline’s, chloramphenicol and fluroquinolones.
Fluoroquinolones block bacterial DNA synthesis by inhibiting bacterial tropoisomerase II (DNA gyres) and tropoisomerase IV. Inhibition of DNA gyres prevents the relaxation of positively supercoiled DNA that is required for normal transcription and replication. Inhibition of tropoisomerase IV interferes with separation of replicated chromosomal DNA into the respective daughter cells during cell division (Betan G. Katzung, 2007) Nalidixic acid, the first member of quinolone, and then newer generation of fluroquinolones discovered to expand the antibacterial spectrum greatly. Newer generation of fluoroquinolones have been obtained by the slight modification of previous generation fluoroquinolones side chain. Fluoroquinolones those commonly used as topical solution are ciprofloxacin, levofloxacin, lomifloxacin, gatifloxacin and moxifloxacin. Their high potency and generally excellent activity against the most frequent gram positive and gram negative ocular pathogens, bactericidal mode of action bioavailability & biocompatibility make fluoroquinolones an excellent initial choice of topical theraphy of bacterial keratitis(C. Stephen Foster, 2005).
In treating patient with ciprofloxacin crystalline white precipitate were observed in the area of epithelial ulceration and this crystalline precipitate reduces the activeconcentration of drug in the stroma at the site of infection(O’ Brien et al,1993). Such crystalline deposition has the potential disadvantage of decreasing visualization of the stromal infiltrate immediately deep to the precipitate for clinical monitoring of the therapeutic progress, there is evidence that ciprofloxacin precipitation may also prevent or delay re-epithelization of a corneal defect(Kanellopoulos AJ et al. 1994). In addition to these unfortunately, their widespread use has lead to emergence of resistance in many bacterial species.
In vitro study indicated that fourth generation fluoroquinolones appear to cover bacterial resistance to the second and third generation fluoroquinolones, and were more potent than the second and third generation fluoroquinolones for gram-positive bacteria, and are equally potent for gram-negative bacteria (Mather R. et al 2002). But the MICs are statistically higher for the second generation fluoroquinolone resistant Staphylococci than for the second generation fluoroquinolone susceptible Staphylococci (Aparna Duggirala et al. 2007). Gatifloxacin 0.3% offers improved activity against Gram-positives, improved activity against atypicals and retained activity against gram-negatives the Gram-positive pathogens, which were resistant to the previous generations of fluoroquinolones, are now susceptible. (Francis S. Mah, M.D. 2004).
Low MICs and higher tissue concentrations are necessary for -effective therapy as well as guarding against antibiotic resistance. Potentially, a million bacteria may exist on the eyelids or in large bacterial infiltrates and abscesses. Bacterial resistance to the second generation fluoroquinolones (ciprofloxacin and ofloxacin) can occur with a single genetic mutation. This means that one bacterium in ten million can develop resistance to a second-generation fluoroquinolone antibiotic. However, the fourth generation fluoroquinolones (moxifloxacin and gatifloxacin) were developed to resist spontaneous mutations that convey antibiotic resistance (Drlica K. A 2001 & Courvalin P, Depardieu F.2000)It generally takes two genetic mutations for resistance to occur with fourth generation fluoroquinolones. This means that one bacterium in ten trillion can develop resistance to fourth-generation fluoroquinolone antibiotics. Even in the instance of ocular infection, a bacterial load of one trillion is not probable to be reached.
A comparison of the in vitro susceptibility patterns and the MICs of gattfloxacin and moxifloxacin (fourth-generation fluoroquinoloncs) with eiprofloxacin and ofloxacin (second-generation fluoroquinolones) and levofloxacin (third-generation fluoroquinolone) using bacterial keratitis isolates was conducted. The fourth-generation fluoroquinolones did, however, demonstrate increased susceptibility for S. aureus isolates that were resistant to ciprofloxacin, levofloxacin, and ofloxacin. The MICs of 8-methoxy fluoroquinolones were statistically lower than the MICs of second-generation fluoroquinolones for all gram-positive bacteria tested.
Furthermore, the fourth generation fluoroquinolones appear to cover second and third generation fluoroquinolone resistance among Staph-ylococcal species (Stroman DW, Clark L, Macke L, Mendoza B, Schlech BA, O’Brien T.2001). The fourth-generation fluoroquinolones did, however, demonstrate increased susceptibility for Staph-ylococcus aureits isolates that were resistant to CIP, LEV and OFX. In general, CIP demonstrated the lowest MICs for gram-negative bacteria. The MICs for fourth-generation fluoroquinolones were statistically lower than the second-generation fluoroquinolones for all gram-positive bacteria tested. Comparing the two fourth-generation fluoroquinolones, MOX demonstrated lower MICs for most gram-positive bacteria, whereas GAT demonstrated lower MICs for most gram-negative bacteria. In conclusion they states that based on in vitro testing, the fourth-generation fluoroquinolones may offer some advantages over those currently available for the treatment of bacterial keratitis.
So in this study we tried to find out a drug that is effective as well as have no adverse effect, can be used as monotheraphy, available, cheap in Bangladesh context.
REVIEW OF LITERATURE
Related previous work:
A study was published in Am J Ophthalmol. 2006; 141(2):282-286, that was conducted by Parmar P; Salman A; Kalavathy CM; Kaliamurthy J; Prasanth DA; Thomas PA; Jesudasan CA in the Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirapalli, India. Their purpose of study to compare the bacteriologic and clinical efficacy of gatifloxacin and ciprofloxacin for the treatment of bacterial keratitis. That was a Prospective, randomized clinical trial. In their study they include total of 104 eyes of 104 patients with bacterial keratitis seen at a tertiary eye-care center. Clinical trial was conducted at the Cornea Service, Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirappalli, India, between April 2004 and March 2005. Patients were assigned in a chronological sequence to one of two masked treatment groups (gatifloxa-cin [GAT] group or ciprofloxacin [CIP] group). Main outcome measure studied was healing of the ulcer. Patients lost to follow-up before complete healing was excluded from further analysis.
They showed that a significantly higher proportion of ulcers in the GAT group exhibited complete healing compared with those in the CIP group (39 eyes [95.1%] vs 38 [80.9%]; P = .042). Gatifloxacin demonstrated a significantly better action than ciprofloxacin against gram-positive cocci in vitro (P < .001), and the percentage of ulcers caused by these pathogens that healed in the GAT group was significantly better than in the CIP group (P = .009). Mean time taken for healing of ulcer and the efficacy against gram-negative bacteria did not significantly differ between the two groups.
In conclusion they states gatifloxacin had a significantly better action against gram-positive cocci both in vitro and in vivo when compared with ciprofloxacin. In view of these organisms being the leading cause of keratitis worldwide, gatifloxacin may be a preferred alternative to ciprofloxacin as the first-line monotherapy in bacterial keratitis.
Another study was conducted by HAROLD JENSEN, Allergan Inc., Irvine, CA, CHAOUKI ZEROUALA LAB Pre-Clinical International Inc., Montreal, Quebec, Canada, MICHEL CARRIER LAB Pre-Clinical International Inc., Montreal, Quebec, Canada, and BRIAN SHORT Allergan Inc., Irvine, CA. published in JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS Volume 21, Number 1, 2005. Their objective was to evaluation of gatifloxacin 0.3% ophthalmic solution efficacy in a corneal ulcer model of Pseudomonas keratitis. In their result all eyes showed evidence of infection by 48 hours postinoculation with 36 of 41 eyes (87.8%) exhibiting moderate-to-severe keratitis. All eyes exhibited corneal healing by day 15, with no significant differences among groups. Three of 4 groups receiving gatifloxacin tended to have smaller fluoresced in retention area scores than did the ciprofloxacin group. No eyes tested positive for Pseudomonas at the end of the study. No corneal precipitates were found following as many as 48 doses/day of gatifloxacin. The most important finding of this study was that gatifloxacin 0.3% ophthalmic solution at the least frequently administered dosing regimen is as effective as ciprofloxacin 0.3%. Other finding is consistent with lower toxicity against cultured human cells of gatifloxacin, compared to ciprofloxacin, especially after exposure to ultraviolet light (Yamamoto, T., Tsurumaki, Y., Takei, M., et al. 2001). In conclusion they state that ophthalmic gatifloxacin 0.3% is at least as effective as ciprofloxacin at healing corneal ulcers infected with Pseudomonas aeruginosa when gatifloxacin is administered less frequently than ciprofloxacin. Trends favored gatifloxacin in fluorescein retention scores.
Another study was conducted by Pragya Parmar MS, Amjad Salman MS, CA Nelson Jesudasan MS and Philip A Thomas MD PhD in the Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirapalli, India & was published in Clinical and Experimental Ophthalmology 2003,- 3 1: 44-47. Their aim to study the dinicai features of pneumococcal keratitis and response to ciprofloxadn therapy.That was a retrospective analysis was undertaken of 58 patients with culture-proven pneumococcal keratitis seen over a period of 2 years. In results they showed that Pneumococcal keratitis accounted for 33.3/6 of bacterial keratitis. Most cases presented with non-severe keratitis (77.5%). Co-existing sac pathology was more frequent in pneumococcal ulcers as compared to non-pneumococcai bacterial ulcers (50% vs 9%, P< 0.001). Characteristic cinicail features enabling an accurate clinical diagnosis were found in 27.5% and lanceolate diplococci on Gram’s stain were identified in 76% of cases. In vitro testing showed a high susceptibility to cephazolin and cipro-floxacin. All patients received ciprofloxadn as first-line therapy. Eighty per cent responded well with complete healing of the ulcer. A second drug was required in 8.5%. They found ciprofloxacin to be effective clinically in treating these ulcers with 80% of ulcers responding well to ciprofloxacin alone. Ciprofloxacin has the added advantage of being commercially available and is thus less prone to contamination or loss of efficacy. It is also more economical. In conclusion they state that ciprofloxacin therapy can be effective in the treatment of pneumococcal keratitis.
M J Bharathi, R Ramakrishnan, R Meenakshi, et al. published in Br J Ophthalmol 2006 90: 1271-1276 Microbiological diagnosis of infective keratitis: comparative evaluation of direct microscopy and culture results showed bacterial pathogens isolated from corneal scrapes of 1151 eyes with infective keratitis treated at a tertiary eye care referral centre in south india.
Table 1. Comparative evaluation of direct microscopy and culture results showed bacterial pathogens isolated from corneal scrapes of 1151 eyes with infective keratitis:
| Bacterial isolates | Pure isolates | Mixed with other bacteria | Mixed with fungal spp | Total no. of bacterial isolates (%) | |
| 1 | Gram-positive cocci | 675 | 65 | 40 | 780(64.14) |
| Streptococcus pneumonia | 417 | 7 | 14 | 438 (36.03) | |
| Staphylococcus epidermidis | 155 | 43 | 24 | 222(18.25) | |
| Staphylococcus aureus | 36 | 10 | 0 | 46 (3.78) | |
| Miccrococcus spp | 6 | 0 | 0 | 6 (0.49) | |
| ?-Haemolytic streptococci | 46 | 5 | 2 | 53 (4.36) | |
| ?-Haemolytic streptococci | 6 | 0 | 6 (0.49) | ||
| Non-haemolytic streptococci | 9 | 0 | 9 (0.74) | ||
| 2 | Gram-positive bacilli | 33 | 22 | 2 | 57 (4.69) |
| Bacillus spp | 12 | 15 | 0 | 27 (2.22) | |
| Corynebacterium spp | 21 | 7 | 2 | 30 (2.47) | |
| 3 | Gram-negative cocci andcoccobacilli | 12 | 12 (0.99) | ||
| Moraxella spp | 9 | 9 (0.74) | |||
| Neisseria spp | 3 | 3 (0.25) | |||
| 4 | Aerobic actinomycetes | 39 | 7 | 46 (3.78) | |
| Nocarcia spp | 39 | 7 | 46 (3.78) | ||
| 5 | Gram-negative bacilli | 245 | 36 | 39 1 | 321 (26.40) |
| Pseudomonas spp | 173 | 29 | 36 1 | 239(19.65) | |
| Enterohacter spp | 26 | 5 | 3 | 34(2.81) | |
| Klehsiella spp | 10 | 2 | 12(0.99) | ||
| Proteus spp | 6 | 6 (0.49) | |||
| Alcaiigens spp | 6 | 6 (0.49) | |||
| Hoemophllus spp | 6 | 6 (0.49) | |||
| Acinetobacter spp | 6 | 6 (0.49) | |||
| E coll | 4 | 4 (0.33) | |||
| Serratia spp | 3 | 3 (0.25) | |||
| Citoobacter spp. | 5 | 5(0.41) | |||
| Total number of isolates (%) | 1004(82.57) | 130 (10.69) | 81 (6.66) | 1216(100) |
In the diagnosis of bacterial keratitis, the sensitivity of Gram stain (100%) obtained in this study was higher than that reported by Sharma S, Kuntmoto DY, Goplnathan U, et al.2002 in early keratitis (36%) and also in advanced keratitis (40.9%). Asbell and Stenson Asbell P, Stenson S.1982 reported 67.0% sensitivity of Gram slain in the detection of bacteria in the US, and Dunlop AA, Wright ED, Howiader SA, et al. 1994 reported 62.0% detection in Bangladesh. The results of this analysis indicate that Gram stain has a vital role in the diagnosis of bacterial keratitis.
Another study published in Am J Ophthalmol 2002; 133:463-466 was conducted by Rookaya Mather MD,Lisa M. Karenchak, BS, [M] SACP ROMANOWSKI, MS, AND REGIS P. KOWALSKI, MS, [M]ASCP with the purpose to show the differences in the susceptibility patterns and the potencies of fourth generation FQs (gatifloxacin-GAT and moxifloxacin-MOX) were compared with third generation (levofloxacin-LEV) and second generation FQs (ciprofloxacin-CIP and ofloxacin-OFX). That was an Experimental laboratory investigation. Their methods was in retrospect, the minimum inhibitory concentrations (MICs) of 93 bacterial endophthalmitis isolates were determined to CIP, OFX, LEV, GAT, and MOX using E-tests. The National Committee of Clinical Laboratory Standards (NCCLS) susceptibility patterns and the potencies of the MICs were statistically compared. Result was with in vitro tests, Staphylococcus aureus isolates that were resistant to CIP and OFX were statistically most susceptible (P = .01) to MOX. Coagulase negative Staphylococci that were resistant to CIP and OFX were statistically most susceptible (P = .02) to MOX and GAT. Streptococcus viridans were more susceptible (P = .02) to MOX, GAT, and LEV than CIP and OFX. Streptococcus pneumoniae was least susceptible (P = .01) to OFX compared with the other FQs. Susceptibilities were equivalent (P = .11) for all other bacterial groups. In general, MOX was the most potent FQ for gram-positive bacteria (P = .05) while CIP, MOX, GAT, and LEV demonstrated equivalent potencies to gram-negative bacteria. Our in vitro study in testing endophthalmitis isolates suggests that the fourth generation fluoroquinolones are more potent than the second and third generation fluoro-quinolones for gram-positive bacteria and are equally as potent for gram-negative bacteria. Furthermore, the fourth generation fluoroquinolones appear to cover second and third generation fluoroquinolone resistance among Staph-ylococcal species (Stroman DW, Clark L, Macke L, Mendoza B, Schlech BA, O’Brien T.2001). In conclusion they states that this in vitro study indicated that fourth generation FQs appear to cover bacterial resistance to the second and third generation FQs, were more potent than the second and third generation FQs for gram-positive bacteria, and are equally potent for gram-negative bacteria. Clinical studies will need to confirm these results.
Stephen V. Scoper Virginia Eye Consultants, Norfolk, Virginia, USA in the study of Review of Third- and Fourth-Generation Fluoroquinolones in Ophthalmology: In- Vivo Efficacy, which was published in Adv Ther. 2008; 25(10); 979-994 states that the five in-vitro studies demonstrated that moxifloxacin and gatifloxacin are statistically more potent than levofloxacin against Gram-positive organisms and similar in potency in most cases of Gram-negative bacteria. In-vivo animal models testing moxifloxacin or gatifloxacin against levofloxacin 0.5% (no clinical trials testing the efficacy of levofloxacin 1.5% have yet been published) demonstrated that fourth- generation agents were superior to third-generation levofloxacin 0.5% for prophylaxis of Gram-positive bacteria-induced infections and were equal to, or better than, levofloxacin 0.5% for the treatment of Gram-negative infections. Fourth-generation agents have increased, potency against Gram-positive bacteria compared with levofloxacin, while maintaining similar potency against Gram-negative bacteria.
Gatifloxacin and Moxifloxacin: An In Vitro Susceptibility Comparison to Levofloxacin, Ciprofloxacin, and Ofloxacin Using Bacterial Keratitis Isolates performed by Kowalski RP, Dhaliwal DK, Karenchak LM, et al. was published in Am J Ophthalmol 2003; 136: 500-505. They compared the in vitro susceptibility patterns and the minimum inhibitory concentrations (MICs) of gatifloxacin (GAT) and moxifloxacin (MOX) (fourth-generation fluoroquinolones) to ciprofloxacin (CIP) and ofloxacin (OFX) (second-generation fluoroquinolones) and levofloxacin (LEV; third-generation flu-oroquinolone) using bacterial keratitis isolates. The goal was to determine whether the fourth-generation fluoroquinolones offer any advantages over the second- and third-generation fluoroquinolones.They found that for most keratitis isolates, there were no susceptibility differences among the five fluoroquinolones. The fourth-generation fluoroquinolones did, however, demonstrate increased susceptibility for Staph-ylococcus aureits isolates that were resistant to CIP, LEV and OFX. In general, CIP demonstrated the lowest MICs for gram-negative bacteria. The MICs for fourth-generation fluoroquinolones were statistically lower than the second-generation fluoroquinolones for all gram-positive bacteria tested. Comparing the two fourth-generation fluoroquinolones, MOX demonstrated lower MICs for most gram-positive bacteria, whereas GAT demonstrated lower MICs for most gram-negative bacteria. In conclusion they states that based on in vitro testing, the fourth-generation fluoroquinolones may offer some advantages over those currently available for the treatment of bacterial keratitis. Clinical studies will be required to confirm these results.
Table 2. Median minimum inhibitory concentrations (MICs; µg/mL) of bacterial keratitis isolates to fluoroquinolones.
| Moxifloxacin | Gatifloxacin | Levofloxacin | Potency by rank | ||
| Bacterial isolates | N | (mox) | (gat) | (lev) | P<0.05 |
| Gram-positive bacteria | |||||
| Staphylococcus aureus FQR | 25 | 1.5 | 4 | 16 | mox>gat>lev |
| Staphylococcus aureus FQS | 25 | 0.032 | 0.094 | 0.19 | mox>gat>lev |
| Coag-neg Staphylococcus FQR | 10 | 2.5 | 3 | 64 | mox=gat>Iev |
| Coag-neg Staphylococcus FQS | 10 | 0.064 | 0.125 | 0.19 | mox>gat>lev |
| Streptococcus pneumonias | 20 | 0.125 | 0.22 | 0.75 | mox>gat>lev |
| Streptococcus viridans | 20 | 0.125 | 0.25 | 0.75 | mox>gat>lev |
| Gram-negative bacteria | |||||
| Pseudomonas aeruginosa FQR | 12 | Resistant to all fluoroquinolones | |||
| Pseudomonas aeruginosa FQS | 25 | 0.5 | 0.25 | 0.38 | gat>lev>mox |
| Serratia rnarcescens | 10 | 0.25 | 0.25 | 0.19 | mox=gat=lcv |
| Haemophilia species | 10 | 0.039 | 0.017 | 0.024 | gat=lev>mox |
| Moraxella species | 10 | 0.047 | 0.032 | 0.047 | gat>mox>lev |
Note: analysis ranked, all MICs from lowest to highest and compared the antibiotics by analysis of variance (ANOVA) of the ranks (not the actual MICs) using Duncan’s multiple comparisons at P <0.05 significance. Coag-neg=coagulase-negative; FQR=fluoroquinolone-resistant (ciprofloxacin and ofloxacin); FQS=fluoroquinolone-sensitivc (ciprofloxacin and ofloxacin).
In the study: Activity of newer fluoroquinolones against gram-positive and gram-negative bacteria isolated from ocular infections: An in vitro comparison conducted by Aparna Duggirala, MSc; Joveeta Joseph, MSc; Savitri Sharma, MD; Rishita Nutheti, MSc; Prashant Garg, MD; Taraprasad Das,MS published in Indian J Ophthalmology 2007;55;15-9 They found that For gram-positive isolates, median MICs of fourth generation fluoroquinolones were lower than second generation. The median MIC was lowest for gatifloxacin and moxifloxacin (0.094 ug/ml) in ciprofloxacin-susceptible isolates of gram-positive bacteria. For ciprofloxacin-susceptible gram-negative bacteria, the median MIC of ciprofloxacin (0.19 ug/ml) was significantly lower than ofloxacin, levofloxacin, gatifloxacin and moxifloxacin (1.5, 0,5, 0.5 and 2 Hg/ml respectively). Ciprofloxacin-resistant isolates of gram-positive bacteria showed higher MIC of levofloxacin, moxifloxacin and gatifloxacin though they remained susceptible to them. None of the fluoroquinolones were effective against ciprofloxacin-resistant gram-negative bacteria. Overall, for gram-positive bacteria, median MICs of levofloxacin, moxifloxacin and gatifloxacin were below ciprofloxacin, the MIC of gatifloxacin and moxifloxacin was equal for gram- positive bacteria. In conclusions: Levofloxacin, gatifloxacin and moxifloxacin are statistically more effective against gram-positive bacteria, the latter two being equally effective. Ciprofloxacin remains the most effective fluoroquinolone against gram-negative bacteria.
CORNEA & Bacterial Corneal Ulcer
We obtain more than 80% of our information from the external world by means of visual function. The cornea serves as the gateway into the eye for external images. The cornea is a transparent avascular tissue that is exposed to the external environment. The anterior corneal surface is covered by the tear film, and the posterior surface is bathed directly by the aqueous humor. The transparent cornea is continuous with the opaque sclera and the semi-transparent conjunctiva. The adult human cornea measures 11 to 12mm horizontally and 10 to 11 mm vertically. It is approximately 0.5 mm thick at the center, and its thickness increases gradually toward the periphery, where it is about 0.7 mm thick. The curvature of the corneal surface is not constant, being greatest at the center and smallest at the periphery. The radius of curvature is between 7.5 and 8.0 mm at the central 3mm optical zone of the cornea where the surface is almost spherical. The refractive power of the cornea is 40 to 44 diopters and constitutes about two-thirds of the total refractive power of the eye. The optical properties of the cornea are determined by its transparency, surface smoothness, contour, and refractive index. Corneal transparency is mostly attributable to the arrangement of collagen fibers in the stroma.
Structure
The structure of the cornea is relatively simple compared with that of other parts of the body. Other avascular tissues of the body include the lens, vitreous body, and components of joints. The cell types that constitute the cornea include epithelial cells, keratocytes (corneal fibroblasts), and endothelial cells. Epithelial cells are derived from the epidermal ectoderm, whereas keratocytes and endothelial cells are of neural crest (neuroectodermal) origin. Cornea is composed of five layers in the microscopic section. They are arranged from before backwards as follows:
Epithelium
The corneal epithelium is composed of nonkeratinized, stratified squamous epithelial cells. The thickness of the corneal epithelium is approximately 50 µm, which is about 10% of the total thickness of the cornea and is constant over the entire corneal surface
The corneal epithelium consists of five or six layers of three different types of epithelial cells: two or three layers of superficial cells, two or three layers of wing cells, and a monolayer of columnar basal cells.
Bowman’s layer
An acellular membrane-like zone known as Bowman’s layer, or Bowman’s membrane, is detectable by light microscopy at the interface between the corneal epithelium and stroma in humans and certain other mammals. Given that this structure, which is 12 µm thick, is not a membrane but rather a random arrangement of collagen fibers and proteoglycans, the term Bowman’s layer is preferable. Bowman’s layer is considered to be the anterior portion of the corneal stroma.
Stroma
The stroma constitutes the largest portion, more than 90%, of the cornea. Many characteristics of the cornea, including its physical strength, stability of shape, and transparency, are largely attributable to the anatomic and biochemical properties of the stroma. The uniform arrangement and continuous slow production and degradation of collagen fibers in the stroma are essential for corneal transparency. The corneal stroma consists of extracellular matrix, keratocytes (corneal fibroblasts), and nerve fibers. This regular arrangement of collagen fibers in the stroma is a major determinant of corneal transparency. Any disturbance in the uniformity of interfiber distance, such as occurs during stromal edema or scarring, can result in a loss of corneal transparency.
Descemet’s membrane
Descemet’s membrane, the basement membrane of the corneal endothelium, gradually increases in thickness from birth (3 µm) to adulthood (8 to 10 µm). Descemet’s membrane is composed mostly of collagen type IV and laminin (Fitch JM, Birk DE, Linsenmayer C et al: 1990) but also contains fibronectin(Suda T, Nishida T, Ohashi Y et ai: 1981 & Fujikawa LS, Foster CS, Harrist TJ et al: 1981) Although tough and resistant to enzymatic degradation by MMPs, Descemet’s membrane is torn easily on exposure to shearing stress. In individuals with certain corneal ulcerations, such as Mooren’s ulcer or bacterial keratitis, Descemet’s membrane remains intact but protrudes as a descemetocele as a result of the intraocular pressure and dissolution of the overlying stroma.. Descemet’s membrane does not regenerate.
Endothelium
A single layer of corneal endothelial cells covers the posterior surface of Descemet’s membrane in a well-arranged mosaic pattern. These cells are uniformly 5 µm thick and 20 µm wide and are polygonal (mostly hexagonal) in shape. In young adults, the cell density is about 3500 cells/mm². Corneal endothelial cells do not proliferate in humans however, demonstrating that they have the ability to undergo mitosis. Factors in aqueous humor or other components of their environment may thus inhibit the proliferation of corneal endothelial cells in situ. Endothelial cell density in the normal, healthy cornea decreases with age (Laule A, Cable MK, Hoffman CE et al: 1978). The most important physiological function of the corneal endothelium is regulation of the water content of the corneal stroma. . The endothelial cells contain ion transport systems that counteract the imbibition of water into the stroma. An osmotic gradient of sodium (Na) is present between the aqueous humor (143 mEq/1) and the stroma (134 mEq/1). This gradient results in the flow of Na from the aqueous humor and in a flux of potassium (K+) in the opposite direction.
Maintenance of Normal Corneal integrity
Maintenance of corneal structure is crucial for the physiological functions of this tissue in refraction and biodefense. Corneal epithelial cells renew rapidly and continuously to maintain the layered structure of the epithelium. The centripetal movement of corneal epithelial cells has been well demonstrated as has the fact that only the basal epithelial cells are capable of proliferation. Epithelial migration is the initial step in the resurfacing of epithelial defects (Binder PS, Wickham MG, Zavala EY et al: 1980). Together with the intracellular cytoskeletal system, signal transduction within cells is important for changes in cell shape and function. Fibronectin provides a provisional matrix during the first phase of epithelial wound healing. Proteolytic enzymes, hyaluronan, growth factors also play an important role in wound healing.
Corneal Function
It performs two major functions. As a component of the body surface, it separates self from the environment and is responsible for protecting the eye from infection and injury. As an optical structure, it provides the majority of the refractive power to the eye and it must remain optically clear and refract light regularly for acute vision.
Infection of the Cornea
Infection of the ocular surface involves four processes: access of the microbe to the ocular surface, attachment of the microbe to the ocular surface, penetration of the microbe through the corneal epithelium, and subsequent growth of the organism. Humans have evolved a robust immune system to prevent and respond to infection. The immune system can be broadly divided into two types. Innate immunity is the first line of defense and includes numerous anatomical, cellular, and biochemical adaptations.
Ulceration
An ulcer is defined as a local epithelial defect with excavation of tissue. There are several mechanisms that contribute to stromal melting and loss. Some of these mechanisms are unique to infectious corneal ulcers. The production of elastase and alkaline phosphates by Pseudomonas and hyaluronidase by Staphylococcus aureus are a few such examples. Other mechanisms of stromal loss are common to ulcers resulting from any etiology. First, breakdown of the corneal epithelium is a prerequisite for development of stromal melting and tissue loss. Several papers have documented that healthy corneal epithelium not only prevents stromal degradation and loss, but also is a prerequisite for stromal healing (Smelser Q: 1960 & Weimar V: 1960) second; most ulcers are associated with a marked inflammatory response. Typically, the inflammatory response is characterized by dense neutrophil infiltration, but other leukocytes play significant roles. The contribution of neutrophils to corneal ulceration has been demonstrated in several animal models. Physical blockade of infiltrating leukocytes in models of corneal injury that induce conical ulceration in control animals prevents ulceration (Kenyon KR et al: 1979). Similarly, systemic depletion of neutrophils can prevent corneal ulceration in guinea pigs (Foster CS et al: 1982). A third and final common mechanism of corneal ulceration is enzymatic degradation of extracellular matrix as part of the normal remodeling of tissues and during tissue repair. The remodeling of tissue involves the degradation and deposition of the local extracellular matrix and is controlled by the release of enzymes by endogenous cells. These enzymes dismantle local extracellular proteins and proteoglycans and the fragments are removed through phagocytosis and degraded by lysosomal hydrolases. New extracellular matrix is then generated.
Epidemiology
The accurate incidence of bacterial keratitis is not known. It is estimated that 30000 cases of microbial keratitis occur in the US annually.3 An estimated 10 to 30 individuals per 100000 contact lens wearers develop ulcerative keratitis annually in the US 4,5 Similar estimates for Great Britain show approximately 1500 annual cases of microbial keratitis from all causes.6 Epidemiological information of developing countries is lacking. Bacterial keratitis is a leading cause of corneal blindness in developing nations.
Principal Causes
There are four principal groups of bacteria that are most frequently responsible (Jones DB. 1979). Micrococcaceaee (Staphylococcus, Micrococcus), the Streptococcus species, the Pseudomonas species, and the Enterobactcriaceae (Citrobtacter, Klebsiella, Enterobacter, Serratia, Proteus). However, virtually any bacteria can potentially cause keratitis under certain favorable conditions. Differences were reported in isolates from patients with supportive keratitis from Ghana and southern India, both of which are at similar tropical latitudes (Leck AK, Thomas PA, Hagan M, et al.2002). There were differences found in the bacterial isolates, with Pseudomonas species the most frequent isolate from Ghana and Streptococcus species the most common isolate from southern India. Geographic variations also exist in the relative frequency of different bacterial organisms as causative agents in keratitis in the United States. Pneumococcus (Streptococcus pneumoniae) was a frequently encountered causative organism of bacterial keratitis in previous clinical reports because of its association with chronic dacryocystitis (Thygeson P: 1948). Pneumococcus has decreased in frequency as a causative organism in developed countries with available effective antibiotics and with refinements in techniques for dacryocystorhinostomy. In developing countries, the pneumococcus remains an important cause of infectious corneal ulceration (Carmichael TR, Wolpert M, Koornhof WJ. 1985 & Srinivasan M, Gonzales CA, George C, et al. 1997). other gram-positive organisms, especially among the Staphylococcus species, continue to be the most commonly isolated causes of bacterial keratitis. Staphylococcus aureus is among the most frequent causative organisms in bacterial keratitis in the Northern and North Eastern United States and Canada, both in normal hosts and in compromised corneas (Asbell P, Stenson LS. 1982). In Great Britain, the most common organisms isolated in bacterial keratitis are Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas and Moraxella. (Coster DJ, Wilhelmus K, Peacock J, et al. 1981).
Risk Factors
Perhaps the most important defense barrier for the cornea is an intact epithelial layer. Most corneal infections result from trauma to the corneal epithelium. Alteration of any of the local or systemic defense mechanisms may also predispose the host to corneal infection. Eyelid abnormalities, including ectropion with exposure, entropion with trichiasis, or lagophthalmos, may be local factors contributing to corneal infection. Abnormalities of the preocular tear film, including aqueous tear layer insufficiency, mucin layer deficiencies from goblet cell loss or dysfunction, and lipid layer instability, may predispose to bacterial keratitis. Lacrimal drainage obstruction may interfere with the lubricating mechanical defense function. The inappropriate use of topical antibiotics could eliminate the natural protection provided by normal ocular flora and predispose to development of opportunistic infections of the cornea. The use of topical corticosteroids can create a localized immunosuppression and present a major risk factor for bacterial keratitis. Corticosteroids prevent neutrophil migration in response to chemotactic factors released during microbial infection (77). Impairment in opsonization is a well-known predisposing factor to infection with encapsulated bacteria, including S. pneumoniae. Streptococcus pyogenes, Haemophilus influenzae, and certain strains of Pseudomonas aeruginosa.
Pathogenesis
The pathogenesis of ocular infectious disease is determined by the intrinsic virulence of the microorganism, the nature of the host response, and the anatomic features of the site of the infection (114). The intrinsic virulence of an organism relates to its ability to invade tissue, resist host defense mechanisms, and produce tissue damage (115). Penetration of exogenous bacteria into the corneal epithelium typically requires a defect in the surface of the squamous epithelial layer. By virtue of specialized enzymes and virulence factors, a few bacteria, such as N. gonorrhoeae, N. meningitidis, C. diphtheriae, Shigella, and Listeria, may directly penetrate corneal epithelium to initiate stromal suppuration.
Many bacteria display several adhesins on fimbriae (pili) and nonfimbriae structures. Such adhesive proteins may recognize carbohydrates on host cells; alternatively, protein-protein interactions can also occur.
Certain bacteria exhibit differential adherence to corneal epithelium. The adherence of S. aureus, S. pneumoniae, and P. aeruginosa to ulcerated corneal epithelium is significantly higher than that of other bacteria and may account in part for their frequent isolation (118).
P. aeruginosa has many virulence factors that contribute to pathogenesis. Cell-associated structures such as pili (119) and flagella (120), and extracellular products, such as alkaline protease (121), elastase (121), exoenzyme S (116), exotoxin A (122), endotoxin (123), slime polysaccharide (124), phospholipase C (121), and leukocidin (121), are associated with virulence, invasiveness, and colonization. Whereas gram-positive bacteria, including S. mirens, adhere to host tissues through fibronectin and collagen (125), P. aeruginosa attaches to cell surfaces that lack fibronectin (126). Bacteria adhere to injured cornea (127), to exposed corneal stroma (128), or to immature nonwounded cornea (129). The corneal epithelial receptors for Pseudomonas species are glycoproteins (130,131).
In addition to organism factors, host lysosomal enzymes and oxidative substances produced by neutrophils, kcratocytcs, and epithelial cells may significantly contribute to the destruction caused by Pseudomonas keratitis (150).
Clinical feature
Once corneal infection is established, there are no absolutely specific clinical symptoms to confirm infection or exclusively distinctive biomicroscopic signs to distinguish the responsible organism(s). Because of the rich innervation of the cornea, the most common symptom of inflammatory lesions of the cornea is pain. Movement of the eyelids over ulcerated corneal epithelium intensifies the pain. Therefore, examination of patients with suspected microbial keratitis is greatly facilitated by instillation of topical anesthetic. Keratitis is usually accompanied by a variable decrease in vision. Reflex tearing, photophobia, and blepharospasm are common and sometime purulent discharge. The conjunctiva may be variably hyperemic and a nonspecific papillary reaction may vary in intensity, depending on the severity of the keratitis. The preocular tear film in bacterial keratitis can be observed by slit-lamp microscopy to contain inflammatory cells and debris. Ipsi-lateral lid edema may be variably observed with bacterial keratitis.
The hallmark clinical signs that are distinctive for suspected infectious keratitis include an ulceration of the epithelium with suppurative stromal inflammation that is either focal or diffuse. Multifocal suppurative inflammation in the cornea is suggestive of mixed infection (polymicrobial keratitis) (169). Polymicrobial keratitis hasbeen observed in from 6% to 56% of overall cases (170,171). Microbial keratitis may occasionally present with an intact epithelium and nonsuppurative multifocal stromal inflammation. The presence of diffuse cellular infiltration in the adjacent stroma and an anterior chamber cellular reaction is highly suggestive for infectious keratitis. The anterior chamber reaction may range from mild flare and cells to severe layered hypopyon formation.
The hypopyon in bacterial keratitis is usually sterile when Descemet’s membrane is intact. Careful measurement and documentation of objective parameters for comparative analysis with subsequent remeasurements are important to monitor the clinical course. Using the adjusting slit beam on the biomicroscope, the overall size of the epithelial involvement can be measured by recording the diameter in two dimensions. Similarly, the area of stromal ulceration can be measured in two meridians. An estimate of the depth of stromal ulceration should be determined by comparing adjacent uninvolved corneal thickness. Slit-lamp photographs are helpful for documentation and monitoring of the clinical course. Initial corneal topographic analysis may be helpful in select cases. Detailed clinical drawings with measurements of the size and depth of infiltration should be recorded at each visit. Additional features to assess include the intensity of suppuration and edema, thickness of the stroma, accompanying scleral suppuration, the degree of anterior chamber and iris inflammation, secondary glaucoma, and the rate of progression or pace of inflammation. A grading system based on the characteristics, including the size of the ulceration in millimeters, percentage depth of ulceration, intensity or density of infiltration, and scleral involvement may provide a guide to the aggressiveness of therapy. More detailed grading systems have been described (174).
Certain characteristic clinical features may be suggestive of specific corneal pathogens, although clinical observation alone should not replace laboratory investigation with corneal scrapings for smears and culture (172,175,176). Gram-positive cocci typically cause localized, round or oval ulcerations with grayish-white stromal infiltrates having distinct borders and minimal surrounding epithelial edema. Staphylococcal keratitis is more frequently encountered in compromised corneas, such as with bullous keratopathy, chronic herpetic keratitis, keratoconjunctivitis sicca, ocular rosacea, or atopic keratoconjunctivitis. With delay in presentation and long-standing infection, both coagulase-positive and coagulase-negative staphylococcal keratitis may cause severe intrastromal abscess and corneal perforation.
After trauma, S. pneumoniae keratitis may present with a deep, oval, central stromal ulceration having serpiginous edges. There is typically dense stromal abscess formation with radiating folds in Descemet’s membrane and moderate accompanying stromal edema. Hypopyon with retrocorneal fibrin deposition is a common clinical feature. Progression to corneal perforation is possible. An abnormal antecedent cornea may modify the classic serpiginous, hypopyon ulcer described after trauma with S, pneumoniae infection. Beta-hemolytic streptococci may cause severe corneal infection with dense suppuration, which may progress to perforation. A distinct, indolent, pauci inflammatory-appearing crystalline keratopathy has been observed in association with streptococcal corneal infection (179-183).
Gram-negative corneal infection typically follows a rapid-paced inflammatory destructive course or, alternatively, a less commonly encountered, slowly progressive indolent ulceration. P. aeruginosa has the most distinctive clinical course after corneal infection. There is a loss of corneal transparency with adjacent peripheral inflammatory epithelial edema and a “ground-glass” stromal appearance.
The typical clinical features of Moraxella keratitis include an indolent corneal ulceration with mild to moderate anterior chamber reaction. The ulceration is usually oval with a predilection for the inferior portion of the cornea.
A rapidly paced, hyper purulent conjunctivitis with marked hyperemia, chemosis, and corneal epithelial ulceration with stromal infiltration should suggest infection with N. gonorrhoeae, or N. meningitidis. A rapid and devastating keratitis may also follow trauma and contamination with B. cereus. B. cereus keratitis corneal infection is characterized by a distinctive stromal ring infiltrate remote from the site of injury with rapid progression to stromal abscess, corneal perforation, and intraocular extension with destruction mediated by specific exotoxins (32). The presence of a distinctive air bubble in the anterior chamber or in the corneal stromal beneath the epithelium, especially after trauma with contaminated soil, should suggest possible infection with spore-forming Clostridium species (42).
Histopathology
Histopathology analysis of bacterial keratitis discloses distinct stages of progressive infiltration, active ulceration, regression, and healing (175). The progressive stage includes adherence and entry of the organism, diffusion of toxins and enzymes, and resultant tissue destruction. Shortly after adherence, polymorph nuclear leukocytes arrive at the corneal wound site (141). Stromal damage from bacterial and neutrophil enzymes facilitates progressive bacterial invasion of the cornea. Penetration into the corneal stroma is accompanied by loss of the bacterial glycocalyx envelope. Initially, the neutrophils arrive in the tear film and enter the cornea through the wound, followed by radial spread through the stroma to the limbus. As infection progresses, limbal vessel ingrowth may deliver neutrophils to the site.
In the second stage, active ulceration, the clinical severity varies with the virulence of the organism and toxin production. There may be progressive-tissue necrosis with subsequent sloughing of the epithelium and stroma, resulting in a sharply demarcated ulcer with a surrounding infiltration of neutrophiis. The necrotic base of the ulcer is surrounded by heaped-up tissue. If organisms penetrate deeper into posterior stroma, progressive keratolysis with stromal thinning may result in descemctocele formation. Corneal perforation may ensue as the next stage.
The third or regressive stage is characterized by an improvement in the clinical signs and symptoms. The natural host defense mechanisms predominate and humoral and cellular immune defenses combine with antibacterial therapy to retard bacterial replication, promote phagocytosis 0f the organism and cellular debris, and halt destruction of stromal collagen.
In the regression phase, a distinct demarcation line may appear as the epithelial ulceration and stromal infiltration consolidate and the edges become rounded. In ulcerative keratitis of long duration, vascularization of the cornea may ensue.
In the final phase or healing stage, the epithelium resurfaces the central area of ulceration and the necrotic stroma is replaced by scar tissue produced by fibroblasts. The reparative fibroblasts are derived from histiocytes and keratocytes that have undergone transformation. Area of stromal thinning may be partially replaced by fibrous tissue. New blood vessel growth directed toward the area of ulceration occurs with delivery of humoral and cellular components to promote further healing. Bowman’s layer does not regenerate, but is replaced with fibrous tissue. New epithelium slowly resurfaces the irregular base. Vascularization gradually disappears, but sometimes a residue of “ghost vessels” remains. The fibrous scar tissue variably produces corneal opacity, although there may be fading of the scar over time with return of relative translucency.
With severe bacterial keratitis, the progressive stage may advance beyond the point where the regressive stage can lead to the healing stage. In such severe ulcerations, stromal keratolysis may progress to corneal perforation with iris prolapse to plug the defect in Descemet’s membrane. Uveal blood vessels may the participate in sealing the perforation, resulting in an adherent vascularized leukoma.
Diagnosis
Based on the presenting clinical history, antecedent risk factors, predisposing ocular and systemic diseases, and distinctive clinical signs, an index of clinical suspicion for infectious keratitis versus a nonmicrobial process is formulated. The timing of clinical presentation may confound the clinician because early in the course it may be difficult to distinguish features of infectious verstis non-infectious corneal processes. Non-infectious ulcerative keratitis may present a clinical dilemma if accompanied by significant corneal inflammation. In patients with longstanding persistent epithelial defects, especially post keratoplasty, stromal infiltration may develop that mimics infectious keratitis. Similarly, individuals with neurotrophic or exposure keratopathy may have ulcerations accompanied by stromal inflammation, which may be indistinguishable from bacterial keratitis. Indolent corneal ulcerations after herpetic keratitis may also resemble the clinical features of infectious corneal ulceration. Particularly difficult to differentiate from early infectious keratitis are the noninfectious immune infiltrates associated with anterior blepharitis or contact lens wear (I 95).
Laboratory Investigation
Clearly, laboratory diagnosis of ocular infection by definitive culturing is the gold standard of clinical management (196). Although it is the preferred approach, microbial culture is often not a practical or a prevailing one for many ophthalmologists (92). Bypassing the step of culturing by opting directly for empirical therapy is a standard office-based approach for some ophthalmologists (197). Obtaining clinical material for laboratory analysis and microbial culture is an important step in the management of suspected infectious keratitis. A standard, thorough methodology should be adopted in all such cases, designed to maximize the yield of recovery of potential corneal pathogens. Knowledge of the likely responsible organisms, including aerobic and anaerobic nonspore-forming bacteria and the possibility of filamentous fungi and yeast, viruses, and protozoa, is important to select the proper laboratory methodology. Standard laboratory procedures can usually recover most organisms by stain or culture (196). In a study assessing the value of Gram stain in management of suppurative keratitis in a developing country, 127 cases of microbial keratitis were examined to determine the relative contributions of Gram stain and culture to diagnosis of the causative organism (199). There were 107 culture-proven cases of microbial keratitis among the 127 patients. Gram stain was positive in 89 cases, which represents 70% of the total and 83% of all culture-proven cases. In 20 cases (16%), no organism was isolated on Gram stain or culture. The results of this study supported the use of both Gram stain and culture in isolation of the causative organisms of suppurative keratitis. With special clinical circumstances, more selective diagnostic techniques and culture media may be indicated. Specimens should be obtained for laboratory microbiologic investigation at the time of presentation immediately after documenting the clinical findings with careful slit-lamp drawings or photography. Clinical material should always be obtained before the initiation of antibiotic treatment. If the patient has been partially treated and the keratin is mild or moderately severe, consideration should be given to suspending antibiotic therapy for a period of 12 hours before return for laboratory investigation. If the keratitis is judged to be severe with a rapid pace of inflammation, specimens should be obtained without delay and antibiotic therapy commenced immediately. Eyelid and conjunctival specimens may be collected for culture and compared with results from corneal culture. The clinical value of eyelid and conjunctival cultures may be limited, however, and even misleading in management of infectious keratiris (196). The most valuable information comes from direct culture of the involved site. Because the cornea may have relatively few infectious organisms compared with other body sites, material from corneal scrapings should be inoculated directly onto the culture media rather than placed into carrier or transport media. Direct plating onto selective media improves the likelihood of recovery, especially with a small number of organisms and potentially fastidious organisms (196). To obtain corneal scrapings comfortably, topical anesthetic is first instilled. Proparacaine hydrochloride 0.5% has the fewest inhibitory effects on organism recovery. Use of tetracaine, cocaine, and other topical anesthetics may significantly reduce organism recovery owing to bacteriostatic effects. A platinum spatula with a rounded flexible tip may be modified with a honing stone to create a narrow-tapered, roughened edge to facilitate removal of corneal material (202). The platinum spatula should be heat sterilized in an alcohol lamp flame and allowed to cool before scraping the cornea. An alternative to the platinum spatula that does not require heat sterilization is the number 15 Bard-Parker (Becton-Dickinson, Franklin Lakes, NJ) surgical blade. The blade is steri