Topical gatifloxacin 0 3% eye drops & ciprofloxacin 0 3% eye drops for the treatment of bacterial

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:  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:  To analyze the symptom and sign in both groups.  To determine the ulcer healing rate both groups.  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 β-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 active concentration 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, In dia, 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 grampositive 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 r esults

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 firstline 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: 12711276 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 1

Gram-positive cocci

Pure Mixed with isolates other bacteria 675 65

Mixed with fungal spp 40

Total no. of bacterial isolates (%) 780(64.14)

Streptococcus pneumonia Staphylococcus epidermidis Staphylococcus aureus Miccrococcus spp ι-Haemolytic streptococci β-Haemolytic streptococci Non-haemolytic streptococci

417 155 36 6 46 6 9

14 24 0 0 2 0 0

438 (36.03) 222(18.25) 46 (3.78) 6 (0.49) 53 (4.36) 6 (0.49) 9 (0.74)

7 43 10 0 5

2 3

4 5

Gram-positive bacilli Bacillus spp Corynebacterium spp Gram-negative cocci and coccobacilli Moraxella spp Neisseria spp Aerobic actinomycetes Nocarcia spp Gram-negative bacilli Pseudomonas spp Enterohacter spp Klehsiella spp Proteus spp Alcaiigens spp Hoemophllus spp Acinetobacter spp E coll Serratia spp Citoobacter spp.

Total number of isolates (%)

33 12 21 12 9 3 39 39 245 173 26 10 6 6 6 6 4 3 5 1004(8 2.57)

22 15 7

7 7 36 29 5 2

130 (10.69)

2 0 2

39 36 3

81 (6.66)

57 (4.69) 27 (2.22) 30 (2.47) 12 (0.99) 9 (0.74) 3 (0.25) 46 (3.78) 46 (3.78) 321 (26.40) 239(19.65) 34(2.81) 12(0.99) 6 (0.49) 6 (0.49) 6 (0.49) 6 (0.49) 4 (0.33) 3 (0.25) 5(0.41) 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 moxifloxacinMOX) 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 fluoroquinolones 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 Gramnegative 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; Âľg/mL) of bacterial keratitis isolates to fluoroquinolones.

Moxiflox Gatifloxacin Levofloxac Potency Bacterial isolates

N

(mox) acin

(gat)

(lev) in

P<0.05 rank

Staphylococcus aureus FQR 25 Staphylococcus aureus FQS 25 Coag-neg Staphylococcus FQR 10

1.5 0.032 2.5

4 0.094 3

16 0.19 64

mox>gat>lev mox>gat>lev mox=gat>Iev

Coag-neg Staphylococcus FQS 10

0.064

0.125

0.19

mox>gat>lev

Streptococcus pneumonias Streptococcus viridans

0.125 0.125

0.22 0.25

0.75 0.75

mox>gat>lev mox>gat>lev

Gram-positive bacteria

20 20

by

Gram-negative bacteria Pseudomonas aeruginosa 12 Pseudomonas aeruginosa FQS 25 FQR Serratia rnarcescens 10

Resistant to all fluoroquinolones 0.5 0.25 0.38

gat>lev>mox

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 gramnegative 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 Âľm, 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 Âľm 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 Âľm) to adulthood (8 to 10 Âľm). 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 ¾m thick and 20 ¾m 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 abnormali ties, 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, proteinprotein 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. Cellassociated 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 coagulasenegative 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 in fection (179-183).

Gram-negative corneal infection typically follows

A

rapid-paced inflammatory

destructive course or, alterna tively, 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 sporeforming Clostridium species (42).

Histopathology Histopathology analysis of bacterial keratitis discloses distinct stages of progressive infiltration, active ulceration, regression, and healing (175). The progressive stage in cludes 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 epithe lium 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 tis sue. 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 â&#x20AC;&#x153;ghost vesselsâ&#x20AC;? 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 cul ture is often not a practical or a prevailing one f o r 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 in flammation, 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 (BectonDickinson, Franklin Lakes, NJ) surgical blade. The blade is sterile in a single-use package.

The rounded tip facilitates obtaining corneal specimens.

Corneal scrapings should be performed along the edge and the base of the ulcerative keratitis lesion. Multiple samples from all areas of the ulceration should be obtained for maximum yield. The selective media agar plates are inoculated by streaking the platinum spatulas lightly over the surface to produce a row of separate inoculation marks in a "C" configuration (C-streaks). Calcium alginate swabs moistened with trypticase soy broth provide another method for collecting corneal specimens (81). A comparative study found a statistically significant higher retrieval rate of bacteria from cases of bacterial keratitis using a moistened calcium alginate swab versus the platinum spatula (171). In cases of deep stromal keratitis, microsurgical scissors, a number 11 Bard-Parker blade (Becton-Dickinson), or a small trephine may be required to sample the cornea adequately. The accompanying intraocular inflammation or layered hypopyon is most often sterile in microbial keratitis, wherein Descemet's membrane remains intact. Obtaining aqueous humor by anterior chamber paracentesis for smear or culture is contraindicaied in most circumstances to avoid the potential risks of inadvertent intraocular inoculation of organisms.

After directly inoculating the choice specimen onto the selective media plates, microscopic slides of corneal scrapings are then made using precleaned glass. The specimens should be placed in the center of the slide over an area of approximately 1 cm in diameter. Pre-etched circles on t he slide allow easy placement of material in a central location for later localization. The microscopic slides should be fixed immediately by immersion in 95% methanol f o r approximately 5 minutes. Heat fixation should be avoided to reduce the likelihood of disrupting the morphology or staining characteristics of the organisms. At least two microscopic slides should be obtained for routine staining and a third should be held for possible special stains.

Stains The Gram stain is the most widely used standard microbiologic stain and its results have been advocated as a guide to the initiation of treatment of bacterial keratitis. Acridine orange is a fluorochromatic dye stain that requires a fluorescent microscope. Gram staining may be simply performed in 5 minutes. After fixation.of the slide in methyl alcohol, the slide is flooded with crystal gentian violet for 1 minute. The slide is then rinsed with water and flooded with Gram's iodine for 1 minute. The slide is again rinsed with water, and then decolorized with acid alcohol for 20 seconds. After rinsing with water, the slide is counterstained by flooding with safranin for 1 minute. The slide is rinsed with water for a final time and then allowed to air or blot dry. Gram stain classifies bacteria into two major groups based on distinct differences in the cell wail.

Gram-positive bacteria retain the gentian violet-iodine complex and appear blue-purple.

Gram-negative bacteria lose the gentian violetâ&#x20AC;&#x201D;iodine complex with the decolonization step and appear pink when counterstained with safranin. The 5-minute Gram stain is

preferred over the 15-second modified stain. If done properly, Gram staining may correctly identify the pathogen in up to 75% of cases caused by a single organism and in 37% of polymicrobial cases (169). The Gram stain was accurate in 61% of cases of bacterial keratitis overall. Slides must be carefully and thoroughly analyzed for correct interpretation. Gram-negative organisms may be more difficult to visualize in corneal scraping specimens than gram-positive organisms. The Giemsa stain may be useful in distinguishing bacteria from fungi. It uses eosin, methylene blue, and azure dyes. With the Giemsa technique, bacteria appear dark blue. Fungal hyphae absorb the stain and generally appear purple or blue. The Giemsa stain identifies normal and inflammatory cells. In addition to bacteria and fungi, identification of chlamydial inclusion bodies and the cysts and trophozoites of Acanthamoeba species may be facilitated with Giemsa stain.

Culture Media Culture on standard bacteriologic media remains the gold standard for diagnosis of suspected bacterial keratitis (196). But the results of corneal cultures should be interpreted with regard to the clinical situation, the adequacy of sampling, and the possibility of contamination by organisms present on the skin, eyelids, and conjunctiva.

Supportive evidence for a pathogenic role of a species is growth on two or more media, heavy growth of the organism, and a Gram stain directly smeared from the lesion containing organisms compatible with those isolated from culture.

Antimicrobial Susceptibility Testing Effective antimicrobial therapy embodies the idea of selective toxicity and requires that the antimicrobial agent reach the site of corneal infection in sufficient concentration to

inhibit and preferably kill the causative microorganism, while causing minimal to no toxicity to the host (226). Several factors modulate the interaction between drug, microorganism, and host. Only the clinical ophthalmologist, having knowledge of the necessary laboratory data, can evaluate the precise interactions and integrate the entire complex in order to initiate a rational therapeutic decision. Standard disk diffusion or microdilution techniques are the preferred laboratory methods for antimicrobial susceptibility testing of bacterial ocular isolates (227â&#x20AC;&#x201D;231).

Corneal Biopsy Corneal trauma may result in inoculation of organisms deep into the stroma. With deep suppurative strornal keratitis, a vertical or oblique incision can allow sampling using a sterile needle or minispatula (196). A sterile 7-0 silk suture may be passed through a deep stromal focus of in fection and then cut into separate sections for inoculation onto specific culture media. Alternatively, a deep lamellar excision can be performed to reach a focal cornea! Abscess (236,237). If there is no access to a sequestered site of deep stromal suppuration, a corneal biopsy may be performed using a disposable skin punch or a small corneal trephine (238-241). The superficial cornea is incised and deepened with a surgical blade (# 11 Bard-Parker) to approximately 0.2 mm. Lamellar dissection is then performed using a sharp blade or microsurgical scissors.

Treatment In general, because of the potential rapid destruction of cornea! tissue that may accompany bacterial keratitis, if there is a clinical suspicion suggestive of a bacterial pathogen, the patient should be treated appropriately for bacterial keratitis until a definitive diagnosis is

established. The objective of therapy in bacterial keratitis is rapidly to eliminate the infective organism, reduce the inflammatory response, prevent structural damage to the cornea, and promote healing of the epithelial surface (3).

The design for drug administration in severe suppurative keratitis includes antibiotics administered frequently, because of the rapid evolution to perforation in keratitis due to virulent pathogens and visual loss secondary to central corneal scarring, many patients with bacterial keratitis having significant ulceration may require hospitalization in the absence of adequate support and assistance.

Antibiotic Therapy The large number of active antimicrobial drugs available to the treating clinician offers the patient with bacterial keratitis a greater chance for cure with less drug-related toxicity while providing alternative choices despite the continuing emergence of drug-resistant pathogenic organisms. Mechanisms of organism resistance include plasmid transfer, either by conjugation or transduction, alteration of permeation, chromosomal mutations, and active efflux. Depending on the mechanism and degree of resistance, the administration of larger dosages of drug that do not cause serious adverse effects may be adequate for clinical cure. With corneal infection, an advantage is direct accessibility through the topical route of administration to achieve high tissue concentrations without significant systemic or local side effects.

Apart from the virulence of the organism, severity of corneal infection is determined in part by the age, genetic makeup, and general health of the patient.

Deficiencies of humoral and cellular defense mechanisms negatively affect patient

response to therapy. Factors that may limit the use of a particular agent in an individual include a reliable history of an allergic reaction to the antibiotic (rash, urticaria, angioedema, wheezing, or anaphylaxis), potential adverse interactions, and predictable adverse effects in certain clinical situations (e.g., use of tetracyclines in young children or pregnant women).

Specific Agents Penicilins After Fleming's announcement of his discovery of peni cillin (250), 10 years elapsed before it was established as a major chemotherapeutic agent (251,252). Interestingly, ophthalmologists performed the first encouraging experiments demonstrating the usefulness of penicillin, even im pure and weak penicillin, in the treatment of pneumococ-cal conjunctivitis in 1932 (253,254). The antimicrobial spectrum of penicillin G for susceptible gram-positive cocci includes S. pyogenes (group A), Streptococcus agalactiae (group B), nonenterococcal group D streptococci (e.g., Streptococcus bovis), viridans streptococci, S. pneumoniae (relatively resistant or absolutely resistant strains to penicillins now exist). Most strains of S. aureus produce beta-lactamase and are resistant.

Penicillin-resistant Neisseria strains, especially penicillinase-producing N. gonorrhoeae, were introduced. Thus, penicillin is no longer recommended for empirical therapy.

Cephalosporins Like the penicillins, cephalosporins contain a bera-lactam ring that is necessary for antibacterial activity. The apparent nucleus of cephalosporin’s is 7-ammocephalosporanic acid (7-ACA). The cephalosporin’s have historically been classified as first-, second-, or third-generation compounds based on their activity against gram-negative bacteria.

First-generation cephalosporin’s were the initial agents developed, having generally excellent gram-positive activity but a narrower gram-negative antibacterial spectrum.

Second-generation cephalospoi INS in general are more active against gram-negative bacteria than first-generation analogs. The third-generation cephalosporins have increased stability to beta-lactamases produced by many gram-negative bacteria. In general, third-generation cephalosporins are less active against gram-positive bacteria than firstgeneration cephalosporin’s (270). Cephalosporins are generally well tolerated, with hyper-sensitivity reactions being the most common systemic adverse effects. They are particularly well tolerated with topical ocular application. But all beta-lactam antibiotics are somewhat unstable in solution, which may limit their activity with topical application for bacterial keratitis (276).

Vancomycin Vancomycin is a glycopeptides antibiotic with activity against penicillin-resistant staphylococci. It is primarily active against gram-positive bacteria and remains one of the most potent antibiotics against 5. Aureus and coagulase-ncgative staphylococci, including methicillin-resistant strains, streptococci (in cluding penicillin G-resistant strains) are highly susceptible to vancomycin. Ototoxicity is the most serious adverse effect of

vancomycin, manifested by auditory nerve damage and hearing loss that may be irreversible

(287). Dose-dependent

nephrotoxicity may occur

with vancomycin

administration, especially with concomitant aminoglycoside therapy (288). Vancomycin is considered the topical therapeutic agent of choice for staphylococcal infections when penicillin or cephalosporins cannot be administered, or if the organism is resistant to these antibiotics (289).

Aminoglycosides Aminoglycosides are principally active against aerobic and facultative gram-negative bacilli. Enterobacteriaceae are usually susceptible, although Salmonella and Shigella species are less susceptible in vivo. Aminoglycosides generally are inactive against anaerobic bacteria, including clostridia and Bacteroides.

Erythromycin Erythromycin has a relatively broad spectrum of activity, especially against most grampositive and some gram-negative bacteria. Most aerobic gram-negative bacilli are resistant to erythromycin. The cell envelopes of most gram-negative bacilli prevent the passive diffusion of erythromycin into the cell.

Fluoroquinolones The most recent class of antibacterial agents to receive PDA approval for the indication of therapy of bacterial keratitis is the fluoroquinolone compounds. The fiuoro-qumolones were serendipitously discovered in 1962, during the purification of

chloroquine. Nalidixic acid, the first member of the quinolone

class. The

fluoroquinolones are rapidly bactericidal in action and exert their effects by variably inhibiting the action of bacterial DNA gyrase, an enzyme essential for bacterial DNA synthesis (301â&#x20AC;&#x201D;303). The commercially available fluoroquinolones (ciprofloxacin, norfloxacin, ofloxacin, gatifloxacin, and moxifloxacin) for topical ophthalmic use have similar antimicrobial spectra that include most aerobic gram-negative and some grampositive bacteria. Although there has been evidence for development of resistance to fluoroquinolones based on in vitro susceptibility testing among ocular isolates, until recently there have-been no clinically significant observations of fluoroquinolone resistance with topical therapy for keratitis. However, clinical cases of bacterial keratitis exhibiting resistance to ciprofloxacin treatment have been reported (324,325). Topical ciprofloxacin (3 mg/mL) and ofloxacin (3 mg/mL) were found to penetrate well into stromal tissue and to be effective in eradicating Pseudomonas organisms compared with controls (327,329). Based on its excellent activity in experimental bactetial ketatitis, topical ciprofloxacin therapy for bacterial keratitis in humans was assessed in an openlabel, nonrandomized clinical trial initially (330). In this noncomparative treatment trial, ciprofloxacin 0.3% topical solution was found to be highly effective in therapy of acute bacterial keratitis and reasonably well tolerated by the ocular surface. Crystalline white ciprofloxacin precipitates were observed in the area of epithelial ulceration in 16% of patients (330). Such crystalline drug precipitation occurs with higher frequency in eyes treated with ciprofloxacin than in those treated with norfloxacin of ofloxacin, consistent with differences in fluoroquinolone compound pH solubility profiles (331). Comparative pharmacokinetic data suggest that this precipitation may reduce the active concenttation of drug in the stroma at the site of infection (332).Such crystalline

deposition has the potential disadvantage of decreasing visualization of the stromal infiltrate immediately deep to the precipitate for clinical monitoring of therapeutic progress. There is evidence that ciprofloxacin precipitation may also prevent or delay reepithelialization of a corneal defect (333). The crystalline corneal precipitates of ciprofloxacin usually spontaneously resolve with cessation of therapy.

Expanded-spectrum fluoroquinolones have a greater activity, especially against grampositive pathogens (338-340). 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 (thirdgeneration fluoroquinolone) using bacterial keratitis isolates was conducted. The fourthgeneration 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. A study to assess the effectiveness of a fourth-generation fluoroquinolone for prophylaxis against multiple drug-resistant staphylococcal keratitis after lamellar keratectomy in a rabbit model was conducted (352). Rabbits underwent unilateral lamellar keratectomy using a manual microkcratome followed by the placement of 1000 colony-forming units of log-phase S. aureus bacteria under each flap. Eyes (seven in each group) were randomized and treated with one of the following agents: sterile balanced salt solution, gatifloxacin (0.3%), ciprofloxacin (0.3%), or levofloxacin (0.5%) immediately and 6, 12, and 18 hours after surgery. Infectious infiltrates developed in five of seven eyes in each group treated with ciprofloxacin, levofloxacin, and balanced salt solution. Gatifloxacin-treated eyes did not

develop clinical infection and exhibited lower mean inflammation scores (p < .01 compared with the other groups). The fourth-generation fluoroquinolone, gatifloxacin, is an effective prophylaxis against the development of keratitis after lamellar keratectomy in rabbits with an organism resistant to methicillin, levofloxacin, and ciprofloxacin (352). In summary, there is considerable experimental and clinical experience with the use of fluoroquinolone solutions for therapy of ocular infections (353). Their high potency and generally excellent activity against the most frequent gram-positive and gram-negative ocular pathogens, bactericidal mode of action, bioavailability, and biocom-patibility make fluoroquinolones an excellent initial choice for the topical therapy of bacterial keratitis.

Routes of Administration One of the fundamental principles of pharmacotherapy is to maximize the amount of drug that reaches the site of action so that sufficient concentrations are achieved to cause a beneficial therapeutic effect (387). Topical application is the mainstay of ocular drug delivery systems and the-topical route is the preferred method of application of an tibiotics in therapy for bacterial keratitis (388-390). Eye-drops are the most common route of antibiotic delivery to the eye. Other topical preparations, including ointments, gels, and sustained-release vehicles, are used to achieve higher concentrations of antibiotics in the corneal stroma.

Drug penetration into the cornea may be increased with higher concentrations, greater lipophilicity, more frequent applications, and enhanced contact time using certain vehicles (391,392). The corneal epithelium represents a potential barrier to antibiotic penetration, and absence of the epithelium, as with ulcerative keratitis, often enhances drug penetration.

Fortified

concentrations

of

antibiotics

are more effective and preferred to the

commercial strength of many antibiotics (388). Fluoroquinolone antibiotics may be effective at their commercial concentrations in therapy for bacterial keratitis given their relatively high potency (25,330,336).

Selection of Antibiotic Therapy The objective for initial antibiotic selection in therapy for bacterial keratitis is rapid elimination of the corneal pathogen. No single topical antibiotic is effective against all potential organisms causing bacterial keratitis. Thus, selection of an antimicrobial agent or agents with a broad spectrum of activity, including the most likely gram-positive and gram-negative corneal pathogens, is desirable. Although penicillin G is superior in activity against S. pneurnoniae and other streptococci, the frequency of penicillinase-producing staphylococci and other organisms requires a penicillinase-resistant agent. Cephalosporins evolved as the drug of choice against unidentified gram-positive cocci. Cefazolin (50 mg/mL) is well tolerated by the ocular surface with topical and subconjunctival routes of administration. In addition, it can be used in therapy of selected patients with a prior history of allergy to penicillin.

Aminoglycosides were the preferred initial antibiotic choice for therapy of suspected gram-negative keratitis. Gentamicin evolved as the initial aminoglycoside agent of choice because of its favorable pharrnacokinetics and excellent activity against Pseudomonas, Kicbsiclla, Enterobacter, and other gram-negative species. With the emergence of gentamicin-resistant strains of P. acrugi-nosa (296), tobramycin has become an alternate initial choice. Approximately 10% of corneal isolates of Pseudomonas may be resistant to aminoglycosides, with some strains being resistant to gcntamicin but sensitive to tobramycin (41 8).

In addition to coverage of gram-positive cocci, the cephalosporins may provide some activity against gram-negative rods. The third-generation cephalosporins provide greater gram-negative coverage, yet have less gram-positive activity than first-generation cephalosporins. Combined therapy with topical

fortified cefazolin (or

another

cephalosporin) and tobramycin (or gentamicin) became the rational initial therapeutic recommendation for polybacterial keratitis or when results of Gram staining were equivocal (3). Aminoglycosides, especially tobramycin, have considerable toxicity when administered topically at frequent intervals for a prolonged period (300).

The fluoroquinolone class of antibiotics possesses potent bactericidal activity against the broad spectrum of gram-negative aerobic bacteria and many gram-positive bacteria, including

penicillinase-producing

and

methicillin-resistant

staphylococci.

The

fluoroquinolones have been shown in several independent clinical trials to provide as safe and effective therapy for acute bacterial keratitis as combination fortified antibiotic treatment (25,336).

The potential role of three topical fluoroquinolones was also evaluated in the treatment of bacterial keratitis by means of a laboratory database (353). Antibiotic susceptibilities were determined for 153 isolates from patients with bacterial keratitis. Results were analyzed tor each fluoroquinolone individually and in combination with cefazolin. Predicted susceptibility to each cefazolinâ&#x20AC;&#x201D;fluoroquinolone combination (98.7%) was superior to that for single-agent therapy with ofioxacin (88.2%), ciprofloxacin (82.3%), or norfloxacin (80.4%; p = .0002). A cefazolin-fluoro-quinolone combination (98.7%) was comparable with a cefazolinâ&#x20AC;&#x201D;gentamicin combination (97.4%). The investigators concluded that combination therapy with cefazolin and a fluoroquinolone offers a reasonable alternative for the

treatment of bacterial keratitis (35.3). Single-agent therapy with fluoroquinolones for visionthreatening bacterial keratitis is not advised.

8-Methoxy fluoroquinolones are designed to provide an expanded spectrum of activity against gram-positive ocular pathogens. In a laboratory study, MICs for gatifloxacm and moxifloxacin were determined in vitro against bacterial strains that were isolated from suspected cases of bacterial keratitis and endophthalmitis (419). The ocular isolates included seven gram-positive, four gram-negative, and three atypical bacterial species.

Gatifloxacin and moxifloxacin exhibited similar activity against six gram-positive organisms: S. epidermidis, S. aureus, S. pneumoniae, S. pyogenes, B, cereus, and E. faecalis. Gatifloxacin demonstrated a broad spectrum of activity against several key ocular pathogens tested in this study, and was at least as effective as moxifloxacin against these pathogens. The fluoroquinolones have been shown to be active against the most important nontuberculous mycobacteria causing keratitis, including some species that are highly resistant to standard antituberculous drugs. M. fortuitum is highly susceptible to ciprofloxacin, levofloxacin, ofloxacin, gatifloxacin, and moxifloxacin (421,422).

Adjunctive Therapy Because of the rich inncrvation of the cornea, ulcerative keratitis is frequently accompanied by significant pain. Pain control with acetammophen or other analgesics may result in improved patient comfort and more effective delivery of the treatment regimen.

Topical cycloplegic agents should be administered to relieve ciliary spasm, alleviate pain, and prevent the formation of synechiae. Topical 0.25% scopolamine or homatropine 5%

used three to four times daily is usually adequate. Significant intraocular inflammation may result in a secondary glaucoma. Elevated intraocular pressure should be monitored and treated with a topical /3-adrenergic blocker or topical or oral carbonic anhydrase inhibitors as required for control. Most patients can be effectively managed as outpatients if there is an adequate support system to allow compliance with the treatment plan.

Patching should be avoided in the initial therapy of bacterial keratitis because this may result in a microenviron-ment favorable to accelerated organism replication. After eradication of the causative bacteria, patching may be applied to assist reepitheliahzation.

Therapeutic soft contact lenses may be a useful adjunct to assist epithelial healing.

Antibiotic administration should continue over the therapeutic soft contact lens. Caution should be exercised because infection may occasionally complicate therapeutic soft contact lens use (424). The therapeutic lens may provide some tectonic support with impending or microscopic corneal perforation.

The precise role and the timing of adjunctive topical corticosteroid use in the therapy of bacterial keratitis are controversial. Reduction in the host inflammatory response, which may contribute to corneal destruction, provides the rationale for corticosteroid use.

Corticosteroids effectively decrease the host inflammatory response initiated by bacterial exotoxins or endotoxins and lytic enzymes released from PMNs. Several experimental studies have failed to demonstrate any deleterious effect from the addition of topical corticosteroids to concomitant bactericidal therapy for bacterial keratitis (430â&#x20AC;&#x201D;432).

Corticosteroids may have a limited role in the therapy of bacterial keratitis to suppress the deleterious effects of inflammation once effective bactericidal therapy has eliminated or reduced the pathogen(s). With gram-positive keratitis, judicious topical application of

corticosteroid may be initiated after several days of intensive, specific antimicrobial therapy.

In confirmed gram-negative infection, or if there is doubt regarding a possible gramnegative coinfec-tion, corticosteroid therapy should be deferred for a longer period of aggressive, specific topical antibiotic therapy. Topical corticosteroids increase the risk of infectious complications affecting the cornea but may or may not have an effect during antibacterial therapy. Given the unproven role of corticosteroids in the adjunctive treatment of bacterial keratitis, a prospective, randomized trial to gather information to provide guidance for this common condition seems justified (438).

Cryotherapy has been applied in experimental animal models with demonstration of bactericidal effects (439). Cryotherapy may be useful in select cases of focal peripheral corneal ulcerations or in Pseudomonas sclerokeratitis (440,441). Caution should be exercised with the administration of Cryotherapy because severe toxic effects may be additive to the keratitis process.

The precise role for adjunctive topical nonsteroidal therapy for bacterial keratitis is not determined. Diclofenac sodium 0.1% therapy did not adversely affect the results of antibiotic therapy with gentamicin 0.3% in the treatment of experimental Pseudomonas keratitis (442). In an experimental model of Pscitdomoints keratitis in rabbits, recurrence was observed in 85% of steroid-treated rabbits versus 12.5% of flurbiprofen-treated rabbits (443). Conversely, in a S. pneumomae model, there were no recurrences experienced, either with steroids or nonsteroidal topical treatment (443).

The application of tissue adhesive (isobtityl cyanoacry-late or orher analogs) has been recommended in progressive stromal keratolysis, thinned dcscemetoceles, or small, perforated infectious ulcerations. The tissue adhesive may have some inherent antibacterial

activity (444). It is toxic to corneal endothelium and thus should be applied only for small perforations. Tissue glue is useful to restore the integrity of the anterior segment and to postpone the need for surgery until antibiotic and antiinflammatory therapy has reduced the ocular inflammation. The edges and bed of the ulceration should be debrided and dried with methylcellulose sponges before the application of tissue adhesive. A therapeutic soft contact lens should be placed over the tissue adhesive to prevent irritation and to protect the glue from mechanical effects of the eyelids.

Excimer laser photoablative treatment of microbial keratitis has been investigated in experimental animal models (446,447). Results of these investigations indicate that advanced stromal keratitis with deep suppuration cannot be eradicated using the excimer laser. Because corneas may be perforated inadvertently during treatment, excimer laser therapy of infectious keratitis should be approached with caution and used only for very select superficial and well-circumscribed lesions.

Corneal patch grafting may be an alternative to the application of tissue adhesives for small corneal perforations resulting from bacterial keratitis. Small partial conjunctival flaps may be used in peripheral ulcerations to assist with healing, but are not recommended for use in impending or perforated central bacterial keratitis. If there is a large perforation or a residual necrotic cornea, a therapeutic penetrating keratoplasty may be indicated (449).

Maximal antibiotic therapy to eradicate the corneal pathogen(s) and to reduce inflammation is recommended before surgery. In addition to topical intensive antibiotic treatment, par-enteral antibiotic therapy should be instituted in the peri-operative period.

The surgeon must select a large trephine to excise completely the area of infection.

Therapeutic penetrating keratoplasty was successful in restoring anatomic and visual results in 75% of grafts performed for bacterial keratitis in one study (450). Complicated cataract may result from severe bacterial keratitis (451). Cataract may result from bacterial toxins, iriclocycliris, and treatment toxicity. Cataract formation may result from severe bacterial keratitis alone, but is probably enhanced by concurrent treatment with high-dose topical corticosteroids. Surgical rehabilitation may require combined cataract extraction with penetrating keratoplasty, depending on the degree of corneal scarring and opacification with cataract formation.

MATERIALS AND METHODS:

Study design

: A prospective, intervention study.

Place of study

: Department of Ophthalmology, Mymensingh Medical College Hospital, Mymensingh. & BNSB, Mymensingh .

Period of study

: July 2008 to October 2009

Sample source

: Sample was taken from patient attended at Eye out patient department of Mymensing Medical College Hospital,,Mymensingh & BNSB, Mymensingh With clinical diagnosis of corneal ulcer.

Same Size

: For study minimum sample size required may be determine by follow by following formula: Sample size n = z2 pq/r2 Here

z = 1.96 for 95% confidence level p = prevalence rate. q=1â&#x20AC;&#x201C;p r = error limit.

As we did not find any prevalence rate of bacterial corneal ulcer in Bangladesh We may consider P = 50% = 0.5 q = 1-p = 1-.5 =.5 r = error limit If we take error limit as 10% of prevalence rate then r = .05 So sample number N

= (1.96)2 x 0.5 x0.5/ (0.05)2 =384

So required sample number is 384. But for time limitation and availability of the patient (Gram stain positive patient with corneal ulcer attended at out patient department of BNSB, Mymensingh was 104 in the last year & no data available from OPD or indoor of MMC&H. So the effective number of sample was reduced to 100.

Selection of cases: Patient with diagnosed bacterial corneal ulcer, age between two years to seventy years and irrespective of sex patients were selected randomly.

Grouping of study patients: Initially lottery was done for determining group. Group A even number & Group B odd number. Every one patient was considered as a single case.

Group A: Patient was treated with topical gatifloxacin 0.3% eye drops.

Group B: Patient was treated with topical ciprofloxacin 0.3% eye drop.

(Group A- 50 Patient & Group B- 50 Patient) Inclusion criteria:



Clinically diagnosed in one eye only of an acute bacterial corneal ulcer of at least 1mm in size .



Presence of bacteria on gram staining.

Exclusion criteria:



Bilateral corneal ulcer or one eyed patient.



Presence of fungi on direct microscopy.



Presence of uncontrolled systemic disease, pregnant or lactating women.



A history of hypersensitivity to fluoroquinolones and related compounds.

Interventions:

After taking proper consent of the patient included in the study the following information’s were recorded in a data base chart: •

History and clinical examination –including general, systemic and Ophthalmological examination.

•

Clinical and laboratory investigations.

History:

Name, age, sex, address, chief complaints, comprehensive medical history including duration of symptoms, known risk factors and predisposing conditions, history of past illness, treatment history & H/O drug allergy.

Clinical examination: Ocular examination were carried out by using – Snellen’s chart, E. chart, Torchlight, Slit lamp biomicroscope, ophthalmoscope etc.

Following points were noted: Visual acuity – Unaided, with pinhole and with lens.

Ocular adnexae particularly eyelids, eyelashes, lacrimal sac regions.

Conjunctiva.

Cornea: Careful measurement and documentation of objective parameters of corneal ulcer.* Anterior chamber.

Iris.

Pupil.

Lens and vetrious condition..

Digital IOP. Fundus by direct ophthalmoscope if needed indirect ophthalmoscope.

All ulcers were scraped for direct microscopy for gram stain & KOH preparation & Culture.

Presence of fungal hyphe or spore seen in KOH preparation was excluded from this study.

The patency of nasolacrimal duct was evaluated by syringing in all cases. *Detail in next page.

Investigation performed for diagnois & Treatment:

•

Gram stain

•

KOH preparation

•

Culture & Sensitivity

Investigation performed for general examination:

•

B.P. examination

•

Urine sugar

•

Blood sugar in selected cases.

Measurement and documentation of objective parameters of corneal ulcer.

Using the adjusting slit beam on the biomicroscope, the overall size of the epithelial involvement was measured by recording the diameter in two dimensions. Similarly, the area of stromal ulceration was measured in two meridians. An estimate of the depth of stromal ulceration was determined by comparing adjacent uninvolved corneal thickness. Slit-lamp photographs was taken for documentation and monitoring of the clinical course. Detailed clinical drawings with measurements of the size of ulcer were recorded at each visit.

Additional features was assessed 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 was recorded. Hypopyon was measured along its vertical height in mm.

Corneal scraping: Corneal scrapings were taken immediately after documenting the clinical findings with careful slit-lamp examination & photography. No. 11 Bard-Parker surgical blade was used.

The blade is sterile in a single-use package. Corneal scrapings were taken along the edge and the base of the ulcer. Multiple samples from all areas of the ulceration were obtained for maximum yield. Microscopic slides of corneal scrapings were then made using precleaned glass and Gram staining may performed. Scraped material was directly plated onto selective media including Blood agar, Chocolate agar, Mac-Conkey agar, Nutrient agar & Thioglycolate broth. Standard disk diffusion techniques were used for antimicrobial susceptibility testing of bacterial ocular isolates.

Treatment and Follow-Up Protocol: Eligable patients were assined in a chronological sequence to one of the two groups. All patients were initially admitted to the hospital and the study medication was instilled hourly until the ulcer began to heal. The patient was then discharge and dosing frequency was gradually reduced. The patient was followed up at weekly intervals and the study medication was continuing until complete healing of the ulcer. Healing defined as closure of the epithelial defect with disappearance of the infiltrate and negative Fluorescein stain.

Ancillary treatment, such as oral analgesics, antiglucoma medications and cycloplegics was continued as required.

Parameters studied:

Symptoms Pain Watering Purulent discharge Redness Photophobia Blurring of vision Signs Area of infiltrate Cilary congestion Size of the ulcer Hypopyon Fluorescein stain •

Visual acuity

•

Growth pattern

•

Sensitivity pattern

•

Outcome of treatment

Grading of symptoms: The symptoms like pain, watering, purulent discharge, redness, photophobia, blurring of vision was followed up & recorded on attendance at OPD, 2 nd, 7th, 14th & 28th day. Symptoms recorded according to grading. The symptoms scored from highest to lowest based on grading according to severity of condition. Subsequent difference of score (whether increase, decrease, same or absent) between two treatment group were studied.

Ocular Pain:

Pain is a subjective sensation that was reported by the patients. The symptoms of ocular pain were given in grade from 0 to 4 based on the severity of finding according to simone et al. (1999) Grade 0

-

No pain.

Grade

1

-

Barely noticeable.

Grade

2

-

Mild pain.

Grade

3

-

Moderate pain.

Grade

4

-

Severe pain.

Watering (grading) 0 = No watering 1 = Watering present but eyelashes are not matted 2 = Eyelashes are matted but no overflow of tear 3 = Eyelashes are severely matted with slight overflow of tear 4 = Constant watering Foreign body sensation (grading) 0 = No foreign body sensation 1 = Mild foreign body sensation 2 = Moderate foreign body sensation 3 = Severe foreign body sensation Purulent discharge (grading) 0 = No 1 = Mild 2 = Moderate 3 = Severe

Photophobia (grading) 0 = Eye can open in sunlight 1 = Difficult to open the eye in sunlight 2 = Eye can open frequently in dim light 3 = Eye can not open in dim light Redness (grading) 0 = No congestion (conjunctiva clear) 1 = Mild congestion 2 = Moderate congestion 3 = Severe congestion Blurring of vision (grading) 0 = No 1 = Mild 2 = Moderate 3 = Severe Grading of signs: Similar to symptoms of the study patients, the signs are area of infiltrate, cilary congestion, size of the ulcer, hypopyon was recorded according to grading. The signs scored from highest to lowest level based on grading according to improvement of their condition. Subsequently the signs analyzed quantitatively find out the mean score statistically significant mean difference of score (whether present or absent) between two treatment groups in terms of visual acuity and pterygium covering the cornea.

Ciliary congestion: Cilary congestion appears as a circumlimbal injection, deep red in colour. The entire conjunctiva may be injected but slit lamp examination reveals marked dilatation and

engorgement of perilimbal vessels. Congestion is due to involvement of anterior ciliary vessels & tributaries. Ciliary congestion were evaluated on a grade 0 to 3 according to kocak et al. (1999).

Grade 0

-

No Congestion.

Grade

1

-

Mild congestion.

Grade

2

-

Moderate congestion.

Grade

3

-

Severe congestion.

Grading on the visual acuity was done according to Abrams, (1995) as follows: For distant 1 – 6/6 2 – 6/9 3 – 6/12 4 – 6/18 5 – 6/24 6 – 6/36 7 – 6/60 8 – 5/60

9 – 4/60 10 – 3/60 11 – 2/60 12 – 1/60 13 – FC ½ m 14 – HM (hand movement) 15 – PL (perception of light) 16 – NPL (No perception of light)

For near 1 – N5 2 – N6 3 – N8 4 – N10 5 – N12 6 – N14 7 – N18 8 – N24

Ethical consideration: The study was approved by the institutional review board.

Patients / Guardians of all patients were informed about the study.

Informed consent was taken from each Patients / guardian.

Data was collected in approved data collection form.

Tools: Pre-tested structured questionnaire, follow-up sheet, slit lamp, microscope, Culture media.

Data collection: By structured questionnaire.

Variables: Demographic variables: •

Age

•

Sex

Clinical Variables: Symptoms •

Pain

•

Watering

•

Purulent discharge

•

Redness

•

Photophobia

•

Blurring of vision

•

Cilary congestion

•

Size of the ulcer

•

Hypopyon

•

Fluorescein stain

•

Visual acuity

•

Growth pattern

Signs

Others

•

Sensitivity pattern

•

Outcome of treatment

Data analysis:

Data analysis was done both manually and by computer. Comparison of data and test of significance was calculated by co-efficient of correlation (r), t- test and (χ)chi-square test.

RATIONALE: Corneal ulcer is a serious sight-threatening condition which can result in permanent loss of vision if appropriate treatment is not instituted promptly. Unfortunately due to emergence of resistance in many bacterial species to many topical antibiotics it became difficult to treat corneal ulcer. Newer fluoroquinolones will overcome this problem.

Gatifloxacin, a fourth generation fluoroquinolone, has shown promise with excellent in vitro activity against most pathogens responsible for occular infections including corneal ulcer. Hitherto, there have been no published studies in Bangladesh comparing gatifloxacin with ciprofloxacin or older fluoroquinolones for the treatment of bacterial corneal ulcer. We aimed to compare the bacteriologic and clinical efficacy of gatifloxacin and ciprofloxacin for the treatment of bacterial corneal ulcer.

RESULTS AND OBSERVATIONS

Initially 100 patients were enrolled in the study but subsequently 12 patients failed to regular follow up during the treatment period were excluded from the study.Then 12 new

patient included. Finally a total of 100 patients were maintained in the study. Fifty of them were treated by the topical gatifloxacin 0.3% eye drops. and fifty patients by the topical ciprofloxacin 0.3% eye drops maintaining the ratio 1:1. Patients were consecutively selected with a view to assess the drug effectiveness of Group A and Group B for the treatment of corneal ulcer.

Table I: age distribution of the patients

Age in years Range (min-max)

Group A (n=50) Mean ±SD 37.5 ±15.4 (8 -70)

Group B (n=50) Mean ±SD 39.9 ±15.9 (1570)

P Value 0.436NS

P value reached from unpaired‘t’ test NS= not significant

Table I showed the age distribution of patients by the pattern of treatment.

Its mean (±SD) was 37.5±15.4 years in Group A and 39.9±15.9 years for group B. No statistically significant difference was found in mean age of the patients (p>0.05).

Table I. Distribution of patients by age and category of treatment

Age in years

Category

of treatment

Total (%)

Gatifloxacin

Ciprofloxacin

<20

n(%) 8(16.0)

n(%) 6(12,0)

14(25.0)

20-29

13(26.0)

13(26.0)

26(25.0)

P v al u e

NS

30-39

15(30.0)

14(28.0)

29(18.3)

40-49

8(16.0)

10(20.0)

18(16.7)

>50

6(12.0)

7(14.0)

13(15.0)

Total

50(100.0)

50(100.0)

100(100.0)

N.B. Figure in parenthesis indicates percentage p value reached from chi square analysis NS = Not significant

A total of 100 patients were consecutively selected with a view to assess the drug effectiveness of cefuroxime and ciprofloxacin for the treatment of corneal ulcer. Table I shows the age distribution of patients by the pattern of treatment. Its mean (±SD) was 37.5±15.4 years in Group A and 39.9±15.9 years for group B. No statistically significant difference was found in age of the patients and pattern of treatment (p>0.05).

Table II: sex distribution of the patients Sex Male Female Total

Group A (n=50) n % 33 66.0 17 34.0 50 100.0

Group B (n=50) n % 31 62.0 19 38.0 50 100.0

P Value NS

P value reached from chi-square test S= Significant NS= Not significan This study was carried out in 100 subjects. They were divided into male and female group. Out of which 17(34.0%) were male and rest 33(66.0%) were female patients

in Group A. In Group B 19(38.0%) were male and rest 31(62.0%) were female patients.

No statistically significant (p>0.05) difference was found between Group A and Group B in chi square test. The results are shown in the table II.

Fig 1: Line graph showing the complaints of the patients during baseline and different follow-up in both groups.

Line graph showed sum of the symptom score during baseline & 1 st to 4th follow up. The drug effect of alleviating the symptom in both groups were decreased on subsequent follow up but more marked in group A .In final follow up the score were almost similar in both groups.

Fig 2: Line graph showed the foreign body sensation of the patients during baseline and different follow-up in both groups.

Line graph showed the foreign body sensation during baseline & 1 st to 4th follow up. The drug effect of alleviating the symptom in both groups were decreased on subsequent follow up but more marked in group A .In final follow up the score were absent in both groups.

Table III: Predisposing factor

Predisposing factor H/O trauma Yes No Total Ocular disease

Group A (n=50) n %

Group B (n=50) N %

P value

40 10 50

38 12 50

0.248

80.0 20.0 100.0

76.0 24.0 100.0

Chronic dacryocystitis Blepharitis Normal Total

3 2 45 50

6.0 4.0 90.0 100.0

2 2 46 50

4.0 4.0 92.0 100.0

0.899

It was observed that 40(80.0%) and 35(70.0%) had history of trauma in group A and group B respectively. Chronic dacryocystitis disease was found 3(6.0%) in group A and 2(4.0%) in group B. Blepharitis was found 2(4.0%) in group A and 2(4.0%) in group B. Table IV: Ciliary congestion of the patients Ciliary congestion Baseline Mild Moderate Severe Total 2nd follow up Nil Mild Moderate Severe Total 3rd follow up Nil Mild Moderate Severe Total 4th follow up Nil Mild Severe Total

Group A (n=50) n %

Group B (n=50) n %

P Value

1 35 14 50

2.0 70.0 28.0 100.0

1 37 12 50

2.0 74.0 24.0 100.0

7 32 9 2 50

14.0 64.0 18.0 4.0 100.0

3 23 19 5 50

6.0 46.0 38.0 10.0 100.0

0.047

34 12 2 2 50

68.0 24.0 4.0 4.0 100.0

24 18 5 3 50

48.0 36.0 10.0 6.0 100.0

0.509

50 0 0 50

100.0 0.0 0.0 100.0

48 2 0 50

96.0 4.0 0.0 100.0

0.901

0.247

Regarding the ciliary congestion of the patients it was found during baseline that in group A Severe 14(28.0%), moderate 35(70.0%), mild 1(02%) and group B Severe 12(24.0%), moderate 37(74.0%), mild 1(02%) respectively.

During 2nd follow up in group A Severe 02(04.0%), moderate 09(18.0%), mild

32(64%) and absent 07(14%) in group B Severe 05(10%), moderate 19(38%), mild 23(46%) and absent 06(12%) P value =0.047.

During 3rd follow up in group a Severe 02(04.0%), moderate 02(04%), mild 12(24%) and absent 34(68%) in group B Severe 03(06%), moderate 05(10%), mild 18(36%) and absent 24(48%) P value =0.509.

In 4th follow up only 02 patient was found mild ciliary congestion in group B, but all Others patients had no ciliary congestion.

Table V: Ulcer of location of the patients

Location of ulcer Central Peripheral Paracentral Total

Group A (n=50) n % 25 50.0 6 12.0 19 38.0 50 100.0

Group B (n=50) n % 22 44.0 9 18.0 19 38.0 50 100.0

P Value 0.673

25(50%) of the ulcer was found in central in group A and 22(44.0%) in group B. Paracentral was found in 19(38.0%) in group A and 19(38.0%) in group B. Peripheral Was found in 6(12.0%) in group A and 9(18.0%) in group B.

Table VI: Distribution of size of the ulcer Size of the ulcer (mm) Baseline <1-2 >2-3 >3-4 >4-6 >6 Total 2nd follow up 0 <1-2 >2-3 >3-4

Group A (n=50) n %

Group B (n=50) n %

7 20 16 7 0 50

14.0 40.0 32.0 14.0 0.0 100.0

9 21 14 5 1 50

18.0 42.0 28.0 10.0 2.0 100.0

14 24 7 3

28.0 48.0 14.0 6.0

5 30 8 4

10 60.0 16.0 8.0

>4-6 >6 Total 3rd follow up 0 <1-2 >2-3 >3-4 >4-6 >6 Total 4th follow up 0 <1-2 >2-3 >3-4 >4-6 >6

2 0 50

4.0 0.0 100.0

2 1 50

4.0 2.0 100.0

40 8 1 0 0 0 50

80.0 18.0 2.0 0.0 0.0 0.0 100.0

36 11 1 1 1 0 50

72.0 22.0 2.0 2.0 2.0 0.0 100.0

50 0 0 0 0 0

100.0 0.0 0.0 0.0 0.0 0.0

50 0 0 0 0 0

100.0 0.0 0.0 0.0 0.0 0.0

It was observed that during baseline most ulcer size was <1-2mm 14(28%) in group A and in group B18(36%), >2-3mm 20(40%) in group A and in group B 21(42%) & >3-4mm 16(32%) in group A and in group B 14(28%). In 2 nd follow up no ulcer was found in 14(28%) in group A and 5(10%) in group B. 1-2mm B ulcer

was found 24(48%) in group A and in group B

30(60%) & >2-3mm 7(14%) in group A and in group B 8(16%). Subsequently ulcer size reduced during 3rd and 4th follow up in both groups.

Table VII: Hypopyon status of the patients

Hypopyon Baseline Present Absent Total 1st follow up Present Absent Total 2nd follow up Present

Group A (n=50) n %

Group B (n=50) n %

22 28 50

44.0 56.0 100.0

17 33 50

34.0 66.0 100.0

0.305

06 44 50

12.0 88.0 100.0

16 34 50

32.0 68.0 100.0

0.048

1

2.0

4

8.0

p

Absent Total

49 50

98.0 100.0

46 50

92.0 100.0

0.181

Table VII shows the status of hypopyon of the patients. In initial presentation Hypopyon was present 22(44%) in group A & 17(34%) in group B. In 1 st follow up it was found that hypopyon was present 06(12%) in group A & 16(32%) in group B. (p=0.048). In 2nd follow up hypopyon was present 01(02%) in group A & 04(08%) in group B. (p=0.181).

Table VIII: Fluorescein stain of the ulcers

Fluorescein stain Base line Positive Negative Total 1st follow up Positive Negative Total 2nd follow up Positive Negative Total 3rd follow up Positive Negative Total 4th follow up Positive Negative Total

Group A (n=50) n %

Group B (n=50) n %

P

50 0 50

100.0 0.0 100.0

50 0 50

100.0 0.0 100.0

42 08 50

84.0 16.0 100.0

48 2 50

96.0 4.0 100.0

36 14 50

72.0 28.0 100.0

45 05 50

90.0 10.0 100.0

0.049

10 40 50

20.0 80.0 100.0

14 36 50

28.0 72.0 100.0

0.348

0 50 50

0.0 100.0 100.0

1 49 50

2.0 98.0 100.0

0.500

-

-

During baseline all patients were found fluorescein stain positive in both groups.

During 1st follow up 84% positive & 16% negative in group A, 96% positive & 04%

negative in groups B. During 2nd follow up 72% positive & 28% negative in group A, 90% positive & 10% negative in group B. During 3rd follow up 20% positive & 80% negative in grous A, 28% positive & 72% negative in group B. In 4th follow up only one patient was found fluorescein stain positive in group B, but all others patients were negative in both groups.

Table IX: Visual outcome in group A and group B

Visual acuity Base line NPL PLPR-HM <1/60-3/60 4/60-6/60 6/36-6/12 6/9-6/6 Total th 4 follow up NPL PLPR-HM <1/60-3/60 4/60-6/60 6/36-6/12 6/9-6/6 Total

Group A (n=50) n %

Group B (n=50) n %

0 12 18 12 8 0 50

0.0 24.0 36.0 24.0 16.0 0.0 100

0 10 14 20 6 0 50

0.0 20.0 28.0 40.0 12.0 0.0 100

1 2 4 13 22 8 50

2.0 4.0 8.0 26.0 44.0 16.0 100.0

2 2 6 8 24 8 50

4.0 4.0 12.0 16.0 48.0 16.0 100.0

Table-IX shows that patient came with PLPR to HM 24.0% and in group A and 20.0% in group B & <1/60 to 3/60 vision 36.0% in group A and 28.0% in group B patient with 4/60 to 6/60 vision 16.0% in group A and 12.0% in group B. During 4 th follow-up better visual acuity of 6/36 to 6/12 was found in 21(42.0%) in Group - A and 24(48.0%) in Group â&#x20AC;&#x201C; B. Table X: Organismâ&#x20AC;&#x2122;s growth pattern following gram stain

Gram stain

Growth of organism

No Growth

Total

Total( group A + group Total( group A + group Gram-positive Gram-negative

B) 63 (33+30) 19 (11+08)

B) 12 (07+05) 06 (02+04)

75 25

Table X: All ulcers that were scraped for direct microscopy with gram stain showed grampositive organism in 75 cases and gram-negative in 25 cases and on culture there was growth of gram-positive organism grow in 63 and gram-negative in 19 cases. Other showed no growth.

Table XI: Clinical (in vivo) response of bacteria Gatifloxacin and Ciprofloxacin therapy Gatifloxacin No. Ulcers

Ciprofloxacin No. Ulcers

Healed n % 32 97.0 12 100.0

Ulcers Total 30 14

Healed n % 21 70.0 9 64.3

ce

Gram- positive Streptococcus pneumoniae

Ulcers Total 33 12

Staphylococcus epidemidis Staphylococcus aureus Bacillus species Gram- negative Pseudomonas aenginosa Enterobacter species Klebsiella pneumoniae Acinetobater species Mixed infections

7 10 4 11 7 1 0 3 0

7 9 4 10 6 1 0 3 0

7 7 2 8 6 0 1 1 2

4 6 2 8 6 0 1 1 2

0.001 NS -

100.0 90.0 100.0 90.9 85.7 100.0 100.0 -

Significan

57.1 85.7 100.0 100.0 100.0 100.0 100.0 100.0

0.001 0.001

Table XI: compares the in vivo clinical response of ulcers to gatifloxacin and ciprofloxacin.

The number of ulcers caused by gram-positive cocci that healed in the group A was 97% & in group B was 70%. When considering individual pathogens, corneal ulcers caused by Streptococcus pneumonia all patient healed with gatifloxacin but 9 out of 14(64.3%) healed with ciprofloxacin. In ulcers caused by Staphylococcus epidermidis 7 out of 7(100%) healed

with gatifloxacin & 4 out of 7(59.1%) healed with ciprofloxacin. In ulcers caused by Staphylococcus aureus 9 out of 10(90%) healed with gatifloxacin & 6 out of 7(85.7%) healed with ciprofloxacin. But corneal ulcers caused by Bacillus species & Gram- negative organisms showed almost similar response.

Table XII: In vitro susceptibility of bacterial isolated from patients with bacterial corneal ulcer No. Isolates Gram- positive Streptococcus pneumoniae Staphylococcus epidemidis Staphylococcus aureus Bacillus species

63 27

17 06

Isolates sensitive to Gatifloxacin n % 62 98.4 26 96.3 13 100.0 17 100.0 06 100.0

Gram- negative Pseudomonas aenginosa Enterobacter species Klebsiella pneumoniae Acinetobater species

19 13

18 12

1 1 4

1 1 4

13

94.7 92.3 100.0 100.0 100.0

Isolates sensitive to Ciprofloxacin n % 51 80.95 20 74.1 9 69.2 16 94.1 6 100.0 17 11 1 1 4

89.5 84.6 100.0 100.0 100.0

Significance

0.001 0.025 0.047 -

Mixed isolate (2) Streptococcus pneumoniae+ Acinetobacter N=1 Streptococcus pneumoniae+ Staphylococcus epidermidis N=1 The microbiological profile of the isolates from culture-positive eyes and the in vitro antibiotic sensitivity pattern of these isolates are showed in Table 13: Among Gram positive organisms, the number of isolates sensitive to gatifloxacin was 62(98.4%) & in ciprofloxacin was 51(80.95%). When considering individual pathogens, Streptococcus pneumoniae 26(96.3) was sensitive to gatifloxacin but 51(74.10%) was sensitive to ciprofloxacin. In Staphylococcus epidermidis 13 out of 13(100%) was sensitive to gatifloxacin but 9 out of 13(69.20%) was sensitive to ciprofloxacin. Staphylococcus aureus 17(100%) was sensitive to gatifloxacin but 16(94.10%) was sensitive to ciprofloxacin. Bacillus species showed similar

response in both group. Among Gram negetive organisms, the number of isolates sensitive to gatifloxacin

was 18(94.7%) & in ciprofloxacin were 17(89.5%). When considering

individual pathogens, Pseudomonas aenginosa 12(94.7%) was sensitive to gatifloxacin but 17(89.5%) was sensitive to ciprofloxacin. Enterobacter species, Klebsiella pneumonia & Acinetobater species showed similar response in both group.

Table XIII: Treatment Failures in Gatifloxacin and Ciprofloxacin Groups

Treatment Group Gatifloxacin

No. of Culture Culture Ulcers Positive Negative Failing to (No. of (No. of Eyes) Heal Eyes) 02 02 00

Ciprofloxacin

09

08

01

Organism Grown

Treatment Required

Pseudomonas aeruginosa (1) Streptococcus pneumonias (1) Staphylococcus epidermidis(3) Streptococcus pneumoniae (4) Staphylococcus aureus (1)

eviscerated (1) healed with other medication (1) eviscerated (2) healed with other medication (6) hooding with tarsorrhaphy (1)

Table XIII: showed treatment failures in Gatifloxacin and Ciprofloxacin group, In group a corneal ulcer of 2 cases fails to heal, both were culture positive (Pseudomonas aeruginosa-1 & Streptococcus pneumonae-1) one needed evisceration and other healed with other medication (topical Moxifloxacin). In group B corneal Ulcer of 8 cases failed to heal, 7 of them were culture positive (Streptococcus Pneumonae-4, Staphylococcus epidermidis -3 & Staphylococcus aureus-1) two Needed evisceration and 6 were healed with other medication (topical Gatifloxacin and Moxifloxacin). One needed hooding with tarsorrhaphy.

Table XIV: Slow response in Gatifloxacin and Ciprofloxacin Groups

Treatment Group

No. of Culture +ve Ulcers (No. of Eyes)

Gatifloxacin

02

01

Ciprofloxacin

06

02

Culture -ve Organism Grown (No. of Eyes) 01 Pseudomonas aeruginosa (1) 04

Streptococcus pneumoniae (2)

Treatment Required Add other medication (1) Tarsorrhaphy (1) Add other medication (2) Tarsorrhaphy (4)

Table XIV: showed slow response in Gatifloxacin and Ciprofloxacin Groups, in group A corneal ulcer of 2 cases showed slow response 1 was culture positive (Pseudomonas aeruginosa-1) other one was culture negative. One needs tarsorrhaphy And other healed with other medication (topical Moxifloxacin). In group B corneal Ulcer of 6 cases showed slow response, 2 were culture positive (Streptococcus Pneumoniae-2) other 4 were culture negative. 4 cases needs tarsorrhaphy and other 2 Were healed with other medication (topical Gatifloxacin and Moxifloxacin).

Discussion Regarding the age distribution it was seen that patient of all age group were affected but the highest numbers of bacterial corneal ulcer were found in the age group between 2039 years. This age group was the adult people and also earning members of their family. They lead and active outdoor life and hence their eye are exposed to ocular trauma and inflection. Similar finding were reported by Wahed and Musch. But in Ghana according to Hagan et al the highest number of bacterial corneal ulcer patients was at age 45 or more. Sex distribution of patients showed a numbers of male and female

patients of bacterial corneal ulcer were 66% and 34% in group A and 62% & 38% in group B table-ll. So the ratio between male and female patients was about 2 : 1 .

According to Srinivasan et al this ration was 1.6:1 in south India but according to Dunlop et al this ratio was 2.2:1 in Bangladesh about fifteen years back. As in Bangladesh and other south Asian countries males are more involved in outdoors activities their susceptibility to ocular trauma is more which can easily lead to bacterial corneal ulcer. This finding also correlates with finding observed Rahman (1981), James et al (1980) Wahed 1981.

In this study the most dominated predisposing ocular condition for developing corneal ulcer was ocular trauma 80.0% in group A and 76.0% in group B (Table-IV). This finding correlates with the finding observed by Rahman (1981) Gomes et al (1983). Though cornea is exposed to surrounding environment and meet to pathogens a delicate balance is exist between cornea and surroundings that help to maintain its integrity.Ocular trauma is such a condition, which creates the breach of epithelium easily and helps invasion of organism and of corneal ulcers. were

The

other

predisposing

condition

Dacryocystitis 3(6.0%) in group A and 2 (4.0%) in group B and Blepharitis 2

(4.0%) in

both group A and in group B. Among the symptoms pain is the most

common as complained by 100% patients of both treatment groups. Pain, redness and decreased visual acuity

were

the most

important

prognostic

index

of

disease progression (Herbert E. Et al 1998).

Pain was due to exposure and irritation of sensory nerve endings of the cornea, due to iritis and also due to increased intraocular pressure produced by hypopyon. Foreign body sensation was an important presentation by all patients. Fig 2: Line graph showed the foreign body sensation during baseline & 1 st to 4th follow up. The drug effect of alleviating

the foreign body sensation in both groups were decreased on subsequent follow up but more marked in group A . But the finding is not statistically significant. In final follow up the score were absent in both groups. Other symptoms presented by different patients were watering, purulent discharge, redness, photophobia, blurring of vision. Fig 1: Line graph showed sum of the symptom score during baseline & 1 st to 4th follow up. The drug effect of alleviating the symptoms

in both groups were decreased on subsequent follow up but

more marked in group A .In final follow up the score were almost similar in both groups.

Photophobia was occurred due to blepharospasm set up by corneal irritation, which became greatly increased on the slightest attempt to separate the lids especially of the attempt was made in bright light. Watering was complained due to reflex secretion of tear following corneal irritation of nerve endings and also irritation of iris by toxins.

Defective vision was due to corneal ulceration, corneal oedema, corneal infiltration, cells and flare of aqueous and hypopyon. Almost all cases were presented with gross visual impairment. Table-IX showed that patient came with PLPR to HM 24.0% and in group A and 20.0% in group B & <1/60 to 3/60 vision 36.0% in group A and 28.0% in group B patient with 4/60 to 6/60 vision 16.0% in group A and 12.0% in group B. This pattern is similar to that reported from populations studied in other countries (Sharma 1981, Dutta et al 1981, Ormerod et al 1986, Foster 1986). During 4th follow-up better visual acuity of 6/36 to 6/12 was found in 21(42.0%) in Group - A and 24(48.0%) in Group â&#x20AC;&#x201C; B. But the finding is not statistically significant.

Regarding clinical signs it was evident that almost all cases were presented with Cilary congestion, corneal ulcer, some with hypopyon and all were fluorescein stain positive.

Regarding the ciliary congestion of the patients it was found during baseline that in group A Severe 14(28.0%), moderate 35(70.0%), mild 1(02%) and group B Severe 12(24.0%),

moderate 37(74.0%), mild 1(02%) respectively. During 2 nd follow up in group A Severe 02(04.0%), moderate 09(18.0%), mild 32(64%) and absent 07(14%) in group B Severe 05(10%), moderate 19(38%), mild 23(46%) and absent 06(12%). It sowed statistically significant decrease of ciliary congestion in 2nd follow up with P value =0.047.

In table V showed 25(50%) of the ulcer was found in central in group A and 22(44.0%) in group B. paracentral was found in 19(38.0%) in group A and 19(38.0%) in group B. peripheral ulcer was found in 6(12.0%) in group A and 9(18.0%) in group B this finding is not statistically significant. In table VI it was observed that during baseline most ulcer size was <1-2mm 14(28%) in group A and in group B 18(36%), >2-3mm 20(40%) in group A and in group B 21(42%) & >3-4mm 16(32%) in group A and in group B 14(28%). No statisticall significance was there. But in 2nd follow up no ulcer was found in 14(28%) in group A and 5(10%) in group B which was statistically significant with P value =0.048.

Table VII shows the status of hypopyon of the patients. In initial presentation hypopyon was present 22(44%) in group A & 17(34%) in group B. In 1 st follow up it was found that hypopyon was present 06(12%) in group A & 16(32%) in group B. Which was statistically significant with P value =0.048.

Table VIII showed during baseline all patients were found fluorescein stain positive in both group. During 2nd follow up 72% positive & 28% negative in group A, 90% positive & 10% negative in group B. Which was statistically significant with P value =0.049.

As we include only those corneal ulcer cases where there is presence of bacteria on gram staining. Table X showed on subsequent culture there was growth of gram-positive organism grow in 63 (group A 33 cases + group B 30 cases) and Gram-negative in 19 (group

A 11 cases + group B 8 cases) cases. Other showed no growth. There was no statistically significant difference between two groups.

Table XI: compares the in vivo clinical response of ulcers to gatifloxacin and ciprofloxacin.

The number of ulcers caused by gram-positive cocci that healed in the group A was 97% & in group B was 70% with p value .001. When considering individual pathogens, corneal ulcers caused by Streptococcus pneumonia all patient healed with gatifloxacin but 9 out of 14(64.3%) healed with ciprofloxacin (p= .001).

In ulcers caused by Staphylococcus

epidermidis 7 out of 7(100%) healed with gatifloxacin & 4 out of 7(59.1%) healed with ciprofloxacin (p= .001). In ulcers caused by Staphylococcus aureus 9 out of 10(90%) healed with gatifloxacin & 6 out of 7(85.7%) healed with ciprofloxacin. But corneal ulcers caused by Bacillus species & Gram- negative organisms showed almost similar response.

The microbiological profile of the isolates from culture-positive eyes and the in vitro antibiotic sensitivity pattern of these isolates are showed in Table 13: Among gram positive organisms, the number of

isolates sensitive to gatifloxacin

was 62(98.4%) & in

ciprofloxacin was 51(80.95%) with p value 001. When considering individual pathogens, Streptococcus pneumoniae 26(96.3) was sensitive to gatifloxacin but 51(74.10%) was sensitive to ciprofloxacin (p= .025). In Staphylococcus epidermidis 13 out of 13(100%) was sensitive to gatifloxacin but 9 out of 13(69.20%) was sensitive to ciprofloxacin (p= .047).

Staphylococcus aureus 17(100%) was sensitive to gatifloxacin but 16(94.10%) was sensitive to ciprofloxacin. Bacillus species showed similar sensitivity pattern in both groups. Among Gram negetive organisms, the number of isolates sensitive to gatifloxacin was 18(94.7%) & in ciprofloxacin were 17(89.5%). When considering individual pathogens, Pseudomonas aenginosa 12(94.7%) was sensitive to gatifloxacin but 17(89.5%) was sensitive to

ciprofloxacin. Enterobacter species, Klebsiella pneumonia & Acinetobater species showed similar response in both groups.

Table XIII: showed treatment failures in Gatifloxacin and Ciprofloxacin group,in group A corneal ulcer of 2 cases fail to heal and in group B corneal ulcer of 8 cases failed to heal, but the sample was to small for significance testing. In group A both were culture positive (Pseudomonas aeruginosa-1 & Streptococcus pneumonae-1) one needed evisceration and other healed with other medication (topical Moxifloxacin). Ingroup B out of 8, 7 of them were culture positive (Streptococcus pneumonae-4, Staphylococcus epidermidis -3 & Staphylococcus aureus-1) two needed evisceration and 6 were healed with other medication (topical Gatifloxacin and Moxifloxacin). One needed hooding with tarsorrhaphy.

Table XIV: showed slow response in Gatifloxacin and Ciprofloxacin Groups, in group A corneal ulcer of 2 cases showed slow response 1 was culture positive (Pseudomonas aeruginosa-1) other one was culture negative. One needs tarsorrhaphy and other healed with other medication (topical Moxifloxacin). In group B corneal ulcer of 6 cases showed slow response, 2 were culture positive (Streptococcus pneumoniae-2) other 4 were culture negative. 4 cases needs tarsorrhaphy and other 2 were healed with other medication (topical Gatifloxacin and Moxifloxacin).

CONCLUSION LIMITATIONS OF STUDY

Limitation of time

:

Study was done from July 2008 to November 2009.

It was limited time for this study.

Financial limitation

:

No Government financial help was present for this study.

Sample size limitation

: 100 patients were selected for the study. If the study was possible in large sample. It will be carried more accurate result.

RECOMMENDATION

 This study should be done in different medical College Hospitals.  Financial support should be allocated by the Government for the study.

 Topical gatifloxacin 0.3% eye drops is more effective than ciprofloxacin 0.3% eye

drops in the treatment of bacterial corneal ulcer.  Chance of bacterial resistance to topical gatifloxacin is much less than the second

generation fluoroquinolones like ciprofloxacin.  So ophthalmologist can use topical gatifloxacin 0.3% eye drops more confidently than

ciprofloxacin 0.3% eye drops in the treatment of bacterial corneal ulcer.