treatment of bacterial cornea

View with images and charts Effect of Topical Gatifloxacin 0.3% & Ciprofloxacin 0.3% in The Treatment Of Bacterial Corneal Ulcer Chapter- 1 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. Stuphen et al. 2007-8). So the corneal health and disease are not less important than that of any vital organ of the body. The cornea has some anatomical and physiological specialties with which it can function without any interruption throughout life. In spite of these specialties the cornea frequently becomes diseased and corneal ulcer is one of the top of the list of corneal disease. So we should give great importance when it becomes diseased.

The avascular, clear anatomical structure of the cornea, with its specialized micro environment 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, Pseudomonas aeruginosa and Enterobactereriace. Uncommon organisms are N. gonorrhoae, N. meningitides, Moraxella species, Haemophilus species, Mycobacteriam spp. & Corynebacteriam spp. (C. Stephen Foster, 2005).

Bacterial corneal ulcer 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 penicillins, cephalosporins, other β-lactum antibiotics, glycopeptides, aminoglycosides, macrolides, tetracyclines, chloramphenicol and fluoroquinolones.

Fluoroquinolones block bacterial DNA synthesis by inhibiting bacterial tropoisomerase II (DNA gyres) and tropoisomerase IV. Inhibition of DNA gyres prevents the relaxation of positively super coiled 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, then newer generation of fluoroquinolones 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 therapy 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, 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 atypical and retained activity against gram-negatives, the gram-positive pathogens, which were resistant to the previous generations of fluoroquinolones, are now susceptible. (Francis S. 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 bacteria 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. 2000). It generally takes two genetic mutations for resistance to occur with fourth generation fluoroquinolones. This means that one bacteria 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 gatifloxacin and moxifloxacin (fourth-generation fluoroquinoloncs) with ciprofloxacin and ofloxacin (secondgeneration 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.

The fourth generation fluoroquinolones did, however, demonstrate increased susceptibility for Staphylococcus aureus 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. 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 for the treatment of bacterial corneal ulcer that is effective as well as have no adverse effect, can be used as mono therapy, available, cheap in Bangladesh context.

Chapter- 2 Justification of the study: 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 excellent in vitro activity against most pathogens responsible for ocular infections including corneal ulcer. Hitherto, there is 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 in the treatment of bacterial corneal ulcer.

Chapter- 3 Hypothesis Topical gatifloxacin 0.3% eye drops is more effective than ciprofloxacin 0.3% eye drops in the treatment of bacterial corneal ulcer.

Chapter- 4 Objectives of this study Objectives: A. General: •

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 find out the symptoms and signs in both groups following treatment.

•

To determine the ulcer healing rate in both groups.

•

To assess the bacteriologic and clinical efficacy of gatifloxacin and ciprofloxacin in the treatment of bacterial corneal ulcer.

Chapter- 5 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. et al: in the Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirapalli, India. 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).

Another study was conducted by Harold Jensen et al; Journal of ocular pharmacology and therapeutics Volume 21, Number 1, 2005. In their result all eyes showed evidence of infection by 48 hours post-inoculation with 36 of 41 eyes (87.8%) exhibiting moderate-tosevere 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 fluorescein 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 et al; in the Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirapalli, India & was published in Clinical and Experimental Ophthalmology 2003,- 3 1: 44-47. They showed that Pneumococcal keratitis accounted for 33.3% of bacterial keratitis. Most cases presented with non-severe keratitis (77.5%). Coexisting sac pathology was more frequent in pneumococcal ulcers as compared to nonpneumococcal bacterial ulcers (50% vs 9%, P< 0.001). Characteristic clinical 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 ciprofloxacin as first-line therapy. Eighty per cent responded well with complete healing of the ulcer. A second drug was required in 8.5%. They found ciprofloxacin to be effective clinically in treating these ulcers with 80% of ulcers responding well to ciprofloxacin alone. Ciprofloxacin has the added advantage of being commercially available and is thus less prone to contamination or loss of efficacy. It is also more economical. In conclusion they state that ciprofloxacin therapy can be effective in the treatment of pneumococcal keratitis.

M J Bharathi, R Ramakrishnan, R Meenakshi, et al. published in Br J Ophthalmol 2006 90: 1271-1276 Microbiological diagnosis of infective keratitis: comparative evaluation of direct microscopy and culture results showed bacterial pathogens isolated from corneal scrapes of 1151 eyes with infective keratitis treated at a tertiary eye care referral centre in south india.

Table 1. comparative evaluation of direct microscopy and culture results showed bacterial pathogens isolated from corneal scrapes of 1151 eyes with infective keratitis:

1

Bacterial isolates

Pure isolates

Gram-positive cocci

675

Mixed with Mixed other with bacteria fungal spp 65 40

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

2 3

4 5

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

417 155 36 6 46 6 9 33 12 21 12

7 43 10 0 5

9 3 39 39 245 173 26 10 6 6 6 6 4 3 5 1004(82.57 )

9 (0.74) 3 (0.25) 7 46 (3.78) 7 46 (3.78) 36 39 321 (26.40) 29 36 239(19.65) 5 3 34(2.81) 2 12(0.99) 6 (0.49) 6 (0.49) 6 (0.49) 6 (0.49) 4 (0.33) 3 (0.25) 5(0.41) 130 (10.69) 81 (6.66) 1216(100)

22 15 7

14 24 0 0 2 0 0 2 0 2

438 (36.03) 222(18.25) 46 (3.78) 6 (0.49) 53 (4.36) 6 (0.49) 9 (0.74) 57 (4.69) 27 (2.22) 30 (2.47) 12 (0.99)

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 stain 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.

Rookaya Mather MD et al: Am J Ophthalmol 2002; 133:463-466 showed the differences in the susceptibility patterns and the potencies of fourth generation FQs (gatifloxacin-GAT and moxifloxacin-MOX) were compared with third generation (levofloxacin-LEV) and second generation FQs (ciprofloxacin-CIP and ofloxacin-OFX). That was an Experimental laboratory investigation. This in-vitro study in testing endophthalmitis isolates suggests that the fourth generation fluoroquinolones are more potent than the second and third generation fluoro-quinolones for gram-positive bacteria and are equally as potent for gram-negative bacteria. Furthermore, the fourth generation fluoroquinolones appear to cover second and third generation fluoroquinolone resistance among Staphylococcal species (Stroman DW et.al. 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 0.5% 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 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 Staphylococcus aureus 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 secondgeneration fluoroquinolones for all gram-positive bacteria tested. Comparing the two fourthgeneration 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.

N

Moxiflox Gatifloxacin Levofloxa Potency acin cin rank (mox) (gat) (lev) P<0.05

25 25 10

1.5 0.032 2.5

4 0.094 3

16 0.19 64

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

10 FQS Streptococcus pneumonias 20 Streptococcus viridans 20

0.064

0.125

0.19

mox>gat>lev

0.125 0.125

0.22 0.25

0.75 0.75

mox>gat>lev mox>gat>lev

Bacterial isolates

by

Gram-positive bacteria Staphylococcus aureus FQR Staphylococcus aureus FQS Coag-neg Staphylococcus FQR Coag-neg Staphylococcus

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

Resistant to all fluoroquinolones 0.5 0.25 0.38

gat>lev>mox

0.25

mox=gat=lcv

0.25

0.19

Haemophilia species

10

0.039

0.017

0.024

gat=lev>mox

Moraxella species

10

0.047

0.032

0.047

gat>mox>lev

Note: analysis ranked, all MICs from lowest to highest and compared the antibiotics by analysis of variance (ANOVA) of the ranks (not the actual MICs) using Duncan's multiple comparisons at p<0.05 significance. Coag-neg = coagulase-negative; FQR = fluoroquinolone-resistant (ciprofloxacin and ofloxacin); FQS = fluoroquinolone-sensitivc (ciprofloxacin and ofloxacin).

In the study: Activity of newer fluoroquinolones against gram-positive and gram-negative bacteria isolated from ocular infections: An in vitro comparison conducted by Aparna Duggirala, MSc; Joveeta Joseph, MSc; Savitri Sharma, MD; Rishita Nutheti, MSc; Prashant Garg,MD; Taraprasad Das, MS published in Indian J Ophthalmology 2007;55;15-9 They found that for gram-positive isolates, median MICs of fourth generation fluoroquinolones were lower than second generation. The median MIC was lowest for gatifloxacin and moxifloxacin (0.094 Âľg/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 Âľg/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 grampositive bacteria, the latter two being equally effective. Ciprofloxacin remains the most effective fluoroquinolone against gram-negative bacteria.

Chapter- 6 Anatomy & Physiology of Cornea 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 towards 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.

Cornea is composed of five layers in the microscopic section. They are arranged from before backwards as follows: Epithelium The corneal epithelium is composed of nonkeratinized, stratified squamous epithelial cells.

The thickness of the corneal epithelium is approximately 50 µm, which is about 10% of the total thickness of the cornea and is constant over the entire corneal surface Bowman's layer An acellular membrane-like zone known as Bowman's layer, or Bowman's membrane, is the interface between the corneal epithelium and 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.

Descemet's membrane Descemet's membrane is the basement membrane of the corneal endothelium. The thickness of this layer is 8 to 10 µm. Descemet’s membrane is composed mostly of collagen type IV and laminin (Fitch J.M. et al: 1990) but also contains fibronectin (Suda T, et al: 1981 & Fujikawa LS, et al: 1981). 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.

Maintenance of Normal Corneal integrity Maintenance of corneal structure is crucial for the physiological functions of this tissue in refraction and bio-defense. 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 et al: 1980).

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.

Chapter- 7 Bacterial Corneal Ulcer

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.

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. The production of elastase and alkaline phosphatase 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 G: 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 corneal 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.

Epidemiology The accurate incidence of bacterial keratitis is not known. It is estimated that 30000 cases of microbial keratitis occur in the US annually (Pepose JS 1992). An estimated 10 to 30 individuals per 100000 contact lens wearers develop ulcerative keratitis annually in the US (MacRae S 1991 & Poggio EC 1989). Similar estimates for Great Britain show approximately 1500 annual cases of microbial keratitis from all causes (Dart JKG 1993).

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 suppurative keratitis from Ghana and southern India, both of which are at similar tropical latitudes (Leck AK, et al.2002).

Risk Factors Perhaps the most important defense barrier for the cornea is an intact epithelial layer. Most corneal infections result from trauma to the corneal epithelium. Alteration of any of the local or systemic defense mechanisms may also predispose the host to corneal infection. Eyelid abnormalities, abnormalities of the preocular tear film, lacrimal drainage obstruction, the

inappropriate use of topical antibiotics & use of topical corticosteroids are major risk factors for bacterial keratitis.

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 (Oâ&#x20AC;&#x2122;Brien TP et al.1996). The intrinsic virulence of an organism relates to its ability to invade tissue, resist host defense mechanisms, and produce tissue damage (Jones DB et al.

1978). 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.

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 (Reichert R 1984).

Bacteria adhere to injured cornea (Hazlett LD, et al.1987), to exposed corneal stroma (Stern GA et al. 1982), or to immature non-wounded cornea (Hazlett LD, et al. 1986). The corneal epithelial receptors for Pseudomonas species are glycoproteins (Hazlett LD, et al. 1992 & Rudner XL, et al. 1992).

In addition to organism factors, host lysosomal enzymes and oxidative substances produced by neutrophils, keratocytcs, and epithelial cells may significantly contribute to the destruction caused by Pseudomonas keratitis (Steuhl KP, et al.1987).

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. 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. Ipsilateral 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. 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.

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 (Liesegang TJ 1988, Ogawa GSH ,1994 & Arffa RC).

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.

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.

Gram-negative corneal infection typically follows a rapid-paced inflammatory destructive course or, alternatively, a less commonly encountered, slowly progressive indolent ulceration.

P. aeruginosa has the most distinctive clinical course after corneal infection. There is a loss of corneal transparency with adjacent peripheral inflammatory epithelial edema and a "groundglass" stromal appearance.

Histopathology Histopathologic analysis of bacterial keratitis discloses distinct stages of progressive infiltration, active ulceration, regression, and healing (Ogawa GSH, et al. 1994). The progressive stage includes adherence and entry of the organism, diffusion of toxins and enzymes, and resultant tissue destruction. In the second stage, active ulceration, the clinical severity varies with the virulence of the organism and toxin production.

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 of 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 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.

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.

Laboratory Investigation Clearly, laboratory diagnosis of ocular infection by definitive culturing is the gold standard of clinical management. Standard laboratory procedures can usually recover most organisms by stain or culture (Wilhelmus KR, et al 1994). With special clinical circumstances, more selective diagnostic techniques and culture media may be indicated. 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. Gram stain classifies bacteria into two major groups based on distinct differences in the cell wall. Gram-positive bacteria retain the gentian violet-iodine complex and appear blue-purple. Gram-negative bacteria lose the gentian violet iodine complex with the decolorization step and appear pink when counterstained with safranin. 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 (Wilhelmus KR, et al 1994).

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 (Amsterdam D.1992). Standard disk diffusion or microdilution techniques are the preferred laboratory methods for antimicrobial susceptibility testing of bacterial ocular isolates (National Committee for Clinical Laboratory Standards, 1993, Neuman MA, et al. 1998 & Sahm DF, et al. 1988).

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 (Wilhelmus KR, et al 1994).

Treatment In general, because of the potential rapid destruction of corneal 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 (Jones DB. 1979).

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.

Specific Agents Penicilins

After Fleming's announcement of his discovery of penicillin (Fleming A.1929), 10 years elapsed before it was established as a major chemotherapeutic agent (Chain E,1940 & Abraham EP, et al.1941). 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 β-lactam ring that is necessary for antibacterial activity. 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 (Lambert HP, et al.1983).

Vancomycin Vancomycin is a glycopeptide antibiotic with activity against penicillin-resistant staphylococci.

Aminoglycosides Aminoglycosides are principally active against aerobic and facultative gram-negative bacilli.

Erythromycin Erythromycin has a relatively broad spectrum of activity, especially against most grampositive and some gram-negative bacteria. .

Fluoroquinolones The most recent class of antibacterial agents to receive FDA approval for the indication of therapy of bacterial keratitis are the fluoroquinolone compounds. The fiuoroquinolones 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 (Smith JT. 1986, Courtright JB,1988 & Hooper DC,1988). 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 gram-positive 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 fluoro-quinolone resistance with topical therapy for keratitis. However, clinical cases of bacterial keratitis exhibiting resistance to ciprofloxacin treatment have been reported (Snyder ME, 1992 & Maffett M, 1993). 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 (O'Brien TP,1988 & Gritz DC,1992). Based on its excellent activity in experimental bactetial ketatitis, topical ciprofloxacin therapy for bacterial keratitis in humans was assessed in an open-label, nonrandomized clinical trial initially (Leibowitz HM, et al.1991). 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 (Leibowitz HM, et al. 1991). 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 (Essepian JP,1995). Comparative pharmacokinetic data suggest that this precipitation may reduce the active concenttation of drug in the stroma at the site of infection (O'Brien TP,1993). 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 (Kanellopoulos AJ, 1994). The crystalline corneal precipitates of ciprofloxacin usually spontaneously resolve with cessation of therapy.

Expanded-spectrum fluoroquinolones have a greater activity, especially against grampositive pathogens (Fugimaki K,1988, Fernandas PB,1988 & Gargallo D, Morris M, Coll R, et al.1988). A comparison of the in vitro susceptibility patterns and the MICs of gatifloxacin 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. 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 (Tungsiripat T, et al.2003). 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 (Tungsiripat T, et al.2003). In summary, there is considerable experimental and clinical experience with the use of fluoroquinolone solutions for therapy of ocular infections . 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 (O'Brien TP, 1995). Topical application is the mainstay of ocular drug delivery systems and the topical route is the preferred method of application of antibiotics in therapy for bacterial keratitis (Shell JW. 1982, Lesar TS, 1985 & Barza M. 1989).

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. Fluoroquinolone antibiotics may be effective at their commercial concentrations in therapy for bacterial keratitis given their relatively high potency (O'Brien TP, 1995, Leibowitz HM, et al. 1991 & Hyndiuk RA, et al.1996).

Selection of Antibiotic Therapy

The objective for initial antibiotic selection in therapy for bacterial keratitis is rapid elimination of the corneal pathogen. The 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.

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 (O'Brien TP, 1995 & Hyndiuk RA, et al.1996).

Adjunctive Therapy Because of the rich innervation of the cornea, ulcerative keratitis is frequently accompanied by significant pain. Pain control with acetaminophen or other analgesics topical cycloplegic agents to relieve ciliary spasm, alleviate pain, and prevent the formation of synechiae.

Elevated intraocular pressure should be monitored and treated with a topical β-adrenergic blocker or topical or oral carbonic anhydrase inhibitors as required for control. After eradication of the causative bacteria, patching may be applied to assist re-epetheliazation.

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

The precise role and the timing of adjunctive topical corticosteroid use in the therapy of bacterial keratitis are controversial. Cryotherapy may be useful in selected cases of focal peripheral corneal ulcerations or in Pseudomonas sclerokeratitis (Codere F, 1981 & Eiferman RA.1979). 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. Excimer laser photoablative treatment of microbial keratitis has been investigated in experimental animal models (Gottsch JD,1991 & Serdarevic O, et al.1985). If there is a large perforation or a residual necrotic cornea, a therapeutic penetrating keratoplasty may be indicated (Kirkness CM, 1991).

Chapter- 8 MATERIALS AND METHODS: 8.1 Research Approach This prospective study was carried out on 112 patients attended at eye out patient department of Mymensingh Medical College Hospital, Mymensingh & BNSB, Mymensingh. All informations were collected in a predesigned structured data collection sheets. Finally we evaluated only 100 patients and they were considered as study sample.

8.2 Study design A prospective study.

8.3 Study population: Patients with clinically diagnosed bacterial corneal ulcer, age between two years to seventy years and irrespective of sex were selected as study population.

8.4 Place of study Department of Ophthalmology, Mymensingh Medical College Hospital, Mymensingh & BNSB, Mymensingh .

8.5 Period of study The study was carried out from 1st July 2008 to 31st December 2009, a period of one year & 6 months.

8.6 Sample size A total number of 100 patients who were clinically diagnosed as bacterial corneal ulcer were included in this study.

Sample size determination: The sample size was determined 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 Ă&#x2014;0.5 Ă&#x2014;0.5/ (0.05)2 =384 So required sample number is 384. But for time limitation, logistic support and availability of the patient the effective number of sample was reduced to 100.

8.7 Sampling method Purposive sampling.

8.8 Ethical consideration: Prior to the commencement of this study, the research protocol was approved by the thesis committee (Local Ethical committee). Objectives of the study along with its procedure, alternative treatment methods, risks and benefits of this study were explained to the patients in easily understandable local language and then informed written consent was taken from each patient/guardian. It was assured that all informed and records would be kept confidential and the procedure was helpful for both the physicians and the patients in making better case management.

8.9 Sample source

Sample was taken from the patients attended at eye out patient department of Mymensingh Medical College Hospital, Mymensingh & BNSB, Mymensingh with clinical diagnosis of corneal ulcer .

8.10 Grouping of patients: Group A: 50 Patients treated with topical gatifloxacin 0.3% eye drops.

Group B: 50 Patients treated with topical ciprofloxacin 0.3% eye drops.

8.11 Inclusion criteria:  Clinically diagnosed an acute bacterial corneal ulcer of at least 1mm in size.

8.12 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.

8.13 Procedure: After taking consent of the patients following informations 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, predisposing factors, history of past illness, treatment and drug history.

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 adenaxae particularly eyelids, eyelashes, lacrimal sac regions.

Conjunctiva.

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

Iris.

Pupil.

Lens and vitreous condition.

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

All ulcer was scraped for direct microscopy for gram stain & KOH preparation & Culture.

Presence of fungal hyphae 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 diagnosis & 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 bio-microscope, 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 was 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 was taken immediately after documenting the clinical findings with careful slit-lamp examination & photography. No. 15/11 Bard-Parker surgical blade was used. The blade is sterile in a single-use package. Corneal scrapings was taken along the edge and the base of the ulcer. Multiple samples from all areas of the ulceration was obtained for maximum yield. Microscopic slides of corneal scrapings were then made using pre-cleaned glass and Gram staining was 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: Eligible patients were assigned in a chronological sequence to one of the two groups. On the date of 1st attending at OPD examination finding was recorded as baseline evaluation. 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 3 rd , 7th, 14th & 28th day 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, anti-glucoma medications and cycloplegics was continued as required.

8.14 Variables: Demographic variables: • Age • Sex Clinical Variables: Symptoms • Pain • Watering • Foreign body sensation • Purulent discharge • Redness • Photophobia • Blurring of vision Signs • Congestion of conjunctiva • Size of the ulcer • Hypopyon • Fluorescein stain Others

• • •

Visual acuity Bacterial growth pattern Drug sensitivity pattern of bacteria.

8.15 Grading of symptoms: The symptoms like pain, watering, foreign body sensation, purulent discharge, redness, photophobia, blurring of vision was followed up & recorded on attendance at OPD, 3 rd , 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 8.16 Grading of signs: Similar to symptoms of the study patients, the signs are area of infiltrate, congestion of conjunctiva, 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.

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 was 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 For near 1 – 6/6 9 – 4/60 1 – N5 2 – 6/9 10 – 3/60 2 – N6 3 – 6/12 11 – 2/60 3 – N8

4 – 6/18 5 – 6/24 6 – 6/36 7 – 6/60 8 – 5/60

12 – 1/60 13 – FC ½ m 14 – HM (hand movement) 15 – PL (perception of light) 16 – NPL (No perception of light)

4 – N10 5 – N12 6 – N14 7 – N18 8 – N24

8.17 Tools: Pre-tested structured questionnaire, follow-up sheet, Snellen chart, 2% Fluroscein stain, slit lamp bio-microscope, microscope, culture media.

8.18 Data analysis: All the data were checked and edited after collection. Then the data were entered into computer and analyzed with the help of SPSS. Chapter- 9 OBSERVATIONS AND RESULTS A total of 100 patients were selected for this study. Fifty of them were treated with gatifloxacin 0.3% eye drops(Group A) and another fifty patients were treated with ciprofloxacin 0.3% eye drops(Group B). All patients were of unilateral eye involvement. The patient was followed up at 3rd, 7th, 14th, 28th day considering 1st, 2nd, 3rd & 4th follow up respectively.

Table I. Distribution of patients by age and category of treatment: Age in years Category of treatment

Pvalue

Group A n(%)

Group B n(%)

<20

8(16.0)

6(12,0)

b

20-29

13(26.0)

13(26.0)

b

30-39

15(30.0)

14(28.0)

b

40-49

8(16.0)

10(20.0)

b

>50 Mean ±SD(Range)

6(12.0) 37.5±15.45(8-70)

7(14.0) 39.9±15.9(15-70)

b

0.563 NS 1.000 NS 0.825 NS 0.602 NS 0.766 NS a 0.436NS

NS = Not significant a p value reached from unpaired ‘t’ test b p value reached from chi square analysis t test perform for mean distribution of table i.e. Mean ±SD Chi square test perform for percentage distribution table Table I shows the age distribution of patients by the pattern of treatment. In group A <20 years ware 8 patients (16%), 20-29 years 13 patients (26%), 30-39 years 15 patients (30%), 40-49 years 8 patients (16%) & >50 years 6 patients (12%). In group B <20 years ware 6

Nu mb er of pat ien ts

patients (12%), 20-29 years 13 patients (26%), 30-39 years 14 patients (28%), 40-49 years 10 patients (20%) & >50 years 7 patients (14%). 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 in between two groups (p= 0.436).

Figure I Bar diagram showing distribution of the patients by age.

Table II: Distribution of the patients by sex.

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 0.676NS

NS= Not significant P value reached from chi-square test Chi-square test perform for percentage distribution table Table II shows sex distribution of enrolled corneal ulcer patients. Out of 50 patient in Group A 33 patients (66.0%) were male and rest 17 patients (34.0%) were female. Out of 50 patient in Group B 31 patients (62.0%) were male and rest 19 patients (38.0%) were female. No statistically significant (p = 0.676) difference was found between Group A and Group B in chi-square test.

Figure II Pie diagram showing distribution of the patients by sex.

Table III: Distribution of patients by predisposing factor.

Predisposing factor H/O trauma Chronic dacryocystitis No known factor Total

Group A (n=50) N % 40 80.0 3 06.0 7 14.0 50 100.0

Group B (n=50) N % 38 76.0 2 04.0 10 20.0 50 100.0

P value a 0.629NS b 0.500NS a 0.424 NS

NS= not significant a p value reached from chi square test b p value reached from fisher exact test Chi-square and fishers test perform for percentage distribution table It was observed that 40 patients (80.0%) and 38 patients (76.0%) had history of trauma in group A and group B respectively. Chronic dacryocystitis was found 3 patients (6.0%) in group A and 2 patients (4.0%) in group B. No known factor was found 7 patients (14.0%) in group A and 10 patients (20.0%) in group B.

Su m of the sy mp to m sco res

Figure III Pie diagram showing distribution of the patients by predisposing factorFigure IV Sum of the symptom scores during baseline evaluation and 1 st, 2nd, 3rd & 4th follow up in Group A & Group B.

D if fe re nt fo ll o Line graph showed sum of the symptomswscore during baseline evaluation & 1 st to 4th follow u during baseline evaluation was 690, in 1 st follow up. In Group A sum of the symptom score nd up that was 588, in 2 follow up 328, in p3rd follow up 195, in 4th follow up 52 & in Group B â&#x2020;&#x2019; evaluation was 675, in 1 st follow up that was 657, sum of the symptom score during baseline nd rd in 2 follow up 430, in 3 follow up 236, in 4th follow up 84. Symptom score were reduced in Group A & Group B on subsequent follow up but more marked in group A .

Table IV: Distribution of patients by congestion of conjunctiva.

Congestion of conjunctiva Group A (n=50) Group B (n=50) P n % n % Value Baseline

Mild Moderate Severe nd 2 follow up Nil Mild Moderate Severe rd 3 follow up Nil Mild Moderate Severe 4th follow up Nil Mild Moderate Severe

1 35 14

2.0 70.0 28.0

1 37 12

02.0 74.0 24.0

7 32 9 2

14.0 64.0 18.0 4.0

3 23 19 5

06.0 46.0 38.0 10.0

34 12 2 2

68.0 24.0 4.0 4.0

24 18 5 3

48.0 36.0 10.0 6.0

49 0 1 0

98.0 0.0 2.0 0.0

48 0 2 0

96.0 00.0 04.0 00.0

a

0.900 NS

a

0.047 S

a

0.220 NS

b

0.500 NS

S= Significant NS= not significant a p value reached from chi square test b p value reached from fisher exact test Chi-square and fishers test perform for percentage distribution table

Nu mb er of pat ien ts

Table IV shows in baseline evaluation congestion of conjunctiva severe in 14 patients(28.0%), moderate in 35 patients(70.0%), mild in 1 patients (02%) in group A and severe in 12 patients(24.0%), moderate in 37 patients(74.0%), mild in 1 patients (02%) in group group B During 2nd follow up in group A severe in 02 patients (04.0%), moderate in 09 patients (18.0%), mild in 32 patients (64%) and absent in 07 patients (14%), in group B severe in 05 patients (10%), moderate in 19 patients (38%), mild in 23 patients (46%) and absent in 06 patients (12%). P value =0.047 which is statistically significant. During 3 rd 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 =0.220). In 4th follow up moderate congestion of conjunctiva was found in 1 patient of group A, 2 patient of group B. (In 1st follow up minimal change or no change occur in congestion of conjunctiva so it was not described above table) Figure V Bar diagram showing distribution of patients by congestion of conjunctiva.

Nu mb er of pat ien ts Nu mb er of pat ien ts Nu mb er of pat ien ts Table V: Distribution of patients by site of corneal ulcer Site of corneal ulcer Central Paracentral Peripheral

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

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

P Value 0.547NS 1.00NS 0.400NS

NS= not significant p value reached from chi square test Chi-square test perform for percentage distribution table Table V showed site of ulcer was central in 25 patients (50%) paracentral was found in 19 patients(38.0%) & peripheral was found in 6 patients (12.0%) in group A.

Central ulcer was found in 22 patients (44.0%), paracentral was found in 19 patients (38.0%) peripheral ulcer was found in 9 patients (18.0%) in group B.

Figure VI Pie diagram showing distribution of patients by site of corneal ulcer Table VI: Distribution of patients by size of the corneal ulcer.

Size of the ulcer (mm) Baseline evaluation 1-2 >2-3 >3-4 >4-6 >6 nd 2 follow up 0 1-2 >2-3 >3-4 >4-6 >6 rd 3 follow up 0 1-2 >2-3 >3-4 >4-6 >6 4th follow up

Group A (n=50) n %

Group B (n=50) n %

7 20 14 7 2

14.0 40.0 28.0 14.0 4.0

9 21 13 5 2

18.0 42.0 28.0 10.0 04.0

14 24 6 3 2 1

28.0 48.0 12.0 6.0 4.0 2.0

5 30 8 4 2 1

10.0 60.0 16.0 08.0 04.0 02.0

39 8 2 0 0 1

78.0 16.0 4.0 0.0 0.0 2.0

36 10 1 0 1 2

72.0 20.0 02.0 0.0 2.0 4.0

P Value

0.957NS

0.373NS

0.7341NS

0 1-2 >2-3 >3-4 >4-6 >6

49 0 0 0 0 1

98.0 0.0 0.0 0.0 0.0 2.0

47 0 1 0 0 2

94.0 0.0 2.0 0.0 0.0 4.0

0.502NS

NS= not significant p value reached from chi square test Chi- square test perform for percentage distribution table It was observed that during baseline evaluation 1-2mm ulcer size was 7(14%), >2-3mm 20(40%) >3-4mm 16(32%), 4-6mm 7(14%) & >6mm 2(4%) in group A and 1-2mm ulcer size was 9(18%), >2-3mm 21(42%) >3-4mm 15(30%), 4-6mm 5(10%) & >6mm 2(4%) in group B. In 2nd follow up no ulcer was found in 14(28%), 1-2mm ulcer size was 24(48%), >2-3mm 6(12%) >3-4mm 3(6%), 4-6mm 2(4%) & >6mm 1(2%) in group A and no ulcer was found in 5(10%),1-2mm ulcer size was 30(60%), >2-3mm 8(16%) >3-4mm 4(8%), 46mm 2(4%) & >6mm 1(2%) in group B.In 3rd follow up no ulcer was found in 39(78%), 12mm ulcer size was 8(16%), >2-3mm 2(4%) >3-4mm 0(0%), 4-6mm 0(0%) & >6mm 1(2%) in group A and no ulcer was found in 36(72%),1-2mm ulcer size was 10(20%), >2-3mm 10(20%), >3-4mm 1(2%), 4-6mm 0(0%), & >6mm 2(4%) in group B. In 4 th follow up 1 patient had ulcer of >6mm in group A and >2-3mm 1(2%), & >6mm 2(4%) in group B.

Num ber of patie nts

Figure VII Bar diagram showing size of the ulcer baseline evaluation & at different follow up

Nu mb er of pat ien ts Nu mb er of pat ien ts

Table VII: Distribution of patients by presence or absence of hypopyon in corneal ulcer Hypopyon

Nu mb er of pat ien ts

Baseline Present Absent 1st follow up Present Absent nd 2 follow up Present Absent rd 3 follow up Present Absent th 4 follow up Present Absent

Group A (n=50) n %

Group B (n=50) n %

p value

22 28

44.0 56.0

17 33

34.0 66.0

a

06 44

12.0 88.0

16 34

32.0 68.0

a

1 49

2.0 98.0

4 46

8.0 92.0

b

1 49

2.0 98.0

3 47

6.0 94.0

b

1 49

2.0 98.0

2 48

4.0 96.0

b

0.305NS

0.015S

0.181NS

0.308NS

0.500NS

S= significant, Ns= not significant a p value reached from chi square test b p value reached from fisher exact test Chi- square and fishers test perform for percentage distribution table Table VII shows the presence or absence of hypopyon of corneal ulcer. In baseline evaluation hypopyon was present 22(44%) in group A & 17(34%) in group B. In 1 st follow up

Nu mb er of pat ien ts

it was found that hypopyon was present 06(12%) in group A & 16(32%) in group B. (p=0.015S). In 2nd follow up hypopyon was present 01(02%) in group A & 04(08%) in group B. (p=0.181).In 3rd follow up hypopyon was present 01(02%) in group A & 03(06%) in group B. (p=0.308).In 4th follow up hypopyon was present 01(02%) in group A & 02(04%) in group B. (p= 0.500).

Figure VIII Bar diagram showing distribution of presence of hypopyon between two groups.

Table VIII: Distribution of patients by fluorescein stain of the ulcers.

Fluorescein stain Group A (n=50) Group B (n=50) P n % n % Value Base line Positive 50 100.0 50 100.0 Negative 00 0.0 00 0.00 st 1 follow up a 0.045S Positive 42 84.0 48 96.0 Negative 08 16.0 02 4.00 2nd follow up a 0.021S Positive 36 72.0 45 90.0 Negative 14 28.0 05 10.0 rd 3 follow up a 0.488NS Positive 11 22.0 14 28.0 Negative 39 78.0 36 72.0 th 4 follow up b 0.308NS Positive 01 2.0 03 6.00 Negative 49 98.0 47 94.0

S= significant, NS= not significant a p value reached from chi square test b p value reached from fisher exact test Chi- square and fishers test perform for percentage distribution table

Nu mb er of pat ien ts

Table VIII shows during baseline all patients were found fluorescein stain positive in both group. During 1st follow up 84% positive & 16% negative in group A, 96% positive & 04% negative in group B. During 2nd follow up 72% positive & 28% negative in group A, 90% positive & 10% negative in group B. During 3 rd follow up 22% positive & 78% negative in group A, 28% positive & 72% negative in group B. In 4 th follow up 2% positive & 98% negative in group A, 06% positive & 94% negative in group B.

Figure IX Bar diagram showing distribution of patients by fluorescein stain positive of the ulcers.

Table IX: Distribution of organismâ&#x20AC;&#x2122;s growth pattern following gram stain.

Gram stain Gram-positive Gram-negative

Group A Growth of No Growth organism 33 02 11 01

Group B Growth of No Growth organism 30 01 08 01

Total 66 21

Table IX: All ulcers that were scraped for direct microscopy with gram stain showed grampositive organism in 66 cases and gram-negative in 21 cases. Rest 13 cases did not show any gram-positive or gram-negative organism. Among gram positive cases there was growth of organism 33 cases, no growth in 02 cases in group A & there was growth of organism 30 cases, no growth in 01 cases in group B, Among 21 gram negative cases there was growth of

Nu mb er of isol ate s

organism 11cases, no growth in 01 cases in group A & there was growth of organism 08 cases, no growth in 01 cases in group B.

Figure X Bar diagram showing distribution of patients by growth pattern organism following gram stain.

Table X: Culture & sensitivity pattern of bacteria isolated from bacterial corneal ulcer No. Isolates sensitive Isolates sensitive Significance Isolates to Gatifloxacin to Ciprofloxacin n % n % a Gram- positive 63 62 98.4 51 80.95 0.001S Streptococcus a pneumoniae 27 26 96.3 20 74.1 0.025S Staphylococcus a epidermidis 13 13 100.0 9 69.2 0.047S Staphylococcus aureus 17 16 94.1 16 94.1 Bacillus species 06 06 100.0 6 100.0 Gram- negative Ps. aeruginosa Enterobacter species Klebsiella pneumoniae Acinetobater species

19 13 1 1 4

18 12 1 1 4

94.7 92.3 100.0 100.0 100.0

17 11 1 1 4

89.5 84.6 100.0 100.0 100.0

S= significant, NS= not significant a p value reached from chi square test b p value reached from fisher exact test Chi- square and fishers test perform for percentage distribution table Mixed isolate(2) Streptococcus pneumoniae+ Acinetobacter N=1

b

0.500NS b 0.500NS -

Nu mb er of isol ate s

Streptococcus pneumoniae+ Staphylococcus epidermidis N=1The microbiological profile of the isolates from culture-positive eyes and the antibiotic sensitivity pattern of these isolates are showed in Table X: 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 27(100.0%) 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 16(94.10%) was sensitive to gatifloxacin & 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 was 17(89.5%). When considering individual pathogens, Pseudomonas aeruginosa 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.

Figure XI Bar diagram showing Culture & sensitivity pattern of bacteria isolated from bacterial corneal ulcer Table XI: Clinical response of bacteria in both groups (Group A =Gatifloxacin and Group B=Ciprofloxacin) Group A No. Ulcers Ulcers Healed Gram- positive

Total 33

n 32

% 97.0

Group B No. Ulcers Ulcers Healed

Significa nce

Total 30

0.003S

n 21

% 70.0

Streptococcus pneumoniae

12

12

Staphylococcus epidemidis

7

7

Staphylococcus aureus

10

9

Bacillus species

4

4

Gram- negative

11

10

Pseudomonas aeruginosa

7

6

Enterobacter species

1

1

Klebsiella pneumonia

0

0

Acinetobater species

3

3

Mixed infections

0

0

100.0

14

9

7

4

7

6

2

2

8

8

6

6

0

0

1

1

1

1

2

2

100.0 90.0 100.0 90.9 85.7 100.0 100.0 -

64.3

0.030S 0.096NS

57.1 85.7 100.0 100.0 100.0 100.0 100.0 100.0

0.669 NS 0.578 NS 0.571 NS -

Nu mb er of pat ien ts

S= significant, Ns= not significant p value reached from fisher exact test Fishers test perform for percentage distribution table Table XI: compares the 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.

Figure XII Bar diagram showing clinical response of bacteria in both groups

Table XII : Failure of therapy by gatifloxacin & ciprofloxacin in both groups.

Treat ment Group Group A

Group B

No. of Ulcers Failing to Heal 02

Culture Positive (No. of Eyes) 02

Culture Organisms Negative Grown (No. of Eyes) 00 Pseudomonas aeruginosa (1) Staphylococcus aureus (1)

09

08

01

Streptococcus pneumoniae (4) Staphylococcus epidermidis(3) Staphylococcus aureus (1)

Complication devloped

Treatment Required

Perforation followed by endophthalmitis (1) Condition detoriating (1) Perforation followed by endophthalmitis (2) Perforation (1) Condition detoriating (6)

Hooding with tarsorrhaphy & intravitrial injection (1) Healed with ciprofloxacin (1) Hooding with tarsorrhaphy & intravitrial injection (2) Hooding with tarsorrhaphy (1) Healed with gatifloxacin (6)

Table XII: showed failure of theraphy in Gatifloxacin and Ciprofloxacin group: In group A corneal ulcer of 2 cases fail to heal, both were culture positive one developed perforation followed by endophthalmitis for which hooding with tarsorrhaphy was done & intravitrial injection was given and other healed with topical ciprofloxacin which was not sensitive to gatifloxacin. In group B corneal ulcer of 9 cases failed to heal, 8 of them were culture positive (Streptococcus pneumonae-4, Staphylococcus epidermidis -3 & Staphylococcus aureus-1) 3 developed perforation 2 of them developed endophthalmitis for which hooding with tarsorrhaphy & intravitrial injection was given and other undergone hooding with tarsorrhaphy and rest 6 were healed with topical gatifloxacin which was not sensitive to ciprofloxacin.

Table XIII: Slow response in Gatifloxacin and Ciprofloxacin Groups Treatment Group Gatifloxacin

No. of Culture +ve Ulcers (No. of Eyes) 02 01

Ciprofloxacin 06

02

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

Treatment Required

04

Tarsorrhaphy (6)

Streptococcus pneumoniae (2)

Tarsorrhaphy (2)

Table XIII: 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. Both underwent tarsorrhaphy.

In group B corneal ulcer of 6 cases showed slow response, 2 were culture positive (Streptococcus pneumoniae-2) other 4 were culture negative. All underwent tarsorrhaphy.

(NB. Slow response means = No significant improvement of symptoms and ulcer remains stationary in 2 consecutive follow up).

Table XIV: Distribution of visual outcome in group A and group B Visual acuity Group A (n=50) Group B (n=50) N % n % Base line NPL 0 0 00.0 00.0 PL-HM 12 10 24.0 20.0 <1/60-3/60 18 20 36.0 40.0 4/60-6/60 12 14 24.0 28.0 6/36-6/12 8 6 16.0 12.0 6/9-6/6 0 0 00.0 00.0 4th follow up NPL 1 2 02.0 04.0 PL-HM 2 2 04.0 04.0 <1/60-3/60 4 6 08.0 12.0 4/60-6/60 13 10 26.0 20.0 6/36-6/12 22 22 44.0 44.0 6/9-6/6 8 8 16.0 16.0

P value

0.866NS

0.951NS

NS= not significant p value reached from chi square test Chi-square test perform for percentage distribution table Table-IX showed that patient came with PL to HM 12 patient (24.0%), <1/60 to 3/60 vision 18 patient (36.0%), 4/60 to 6/60 vision 12 patient(24.0%), 6/36-6/12 vision in 8 patient(16.0%)and in group A and PL to HM 10 patient(20.0%), <1/60 to 3/60 vision 20 patient(40.0%), 4/60 to 6/60 vision 14 patient(28.0%), 6/36-6/12 vision in 6 patient(12.0%)and in group B. During 4th follow-up NPL 1 patient, PL to HM in 2 patients (4.0%), <1/60 to 3/60 vision 4 in patients (8.0%), 4/60 to 6/60 vision in 13 patients(26.0%), 6/36-6/12 vision in 22 patients(44.0%) 6/9-6/6 vision in 8 patients (16.0%) in group A and NPL 2 patient, PL to HM in 2 patients (4.0%), <1/60 to 3/60 vision 6 in patients (12.0%), 4/60 to 6/60 vision in 10 patients(20.0%), 6/36-6/12 vision in 22 patients(44.0%) 6/9-6/6 vision in 8 patients (16.0%) in group B. Figure XIII Bar diagram showing distribution of visual outcome in group A and group B

Nu mb er of pat ien ts Nu mb er of pat ien ts

V is u al ac ui ty

V is u al ac ui Figure XIV Patient treated with gatifloxacin ty

Baseline evaluation th 2nd 4follow follow upup

1st follow up 3rd follow up

Figure XV Patient treated with ciprofloxacin 1.

Baseline evaluation

2nd follow up

1st follow up

3rd follow up

4th follow up

Chapter- 10 Discussion 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, Pseudomonas aeruginosa and enterobactereriace. (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 to eliminate the infective organism rapidly, to reduce the inflammatory response, to prevent structural damage to the cornea and to promote healing of the epithelial surface (Jones DB.1979). A large number of active antimicrobial drugs available for the treatment of bacterial corneal ulcer have 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). The newer generation of fluoroquinolones are likely to play an important role in the treatment of bacterial keratitis in the future because of an increasing resistance to the second-generation fluoroquinolones that are in current use. In vitro studies on isolates from bacterial infections of the eye have shown an encouraging response to gatifloxacin, including those organisms resistant to ciprofloxacin.

(Mather R et al: 2002; & Tungsiripat T,et al: 2003). These results had been validated by clinical trials on human eyes (Parmar P et al: 2006) . 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 bacteria 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 2000). It generally takes two genetic mutations for resistance to occur with fourth generation fluoroquinolones. This means that one bacteria 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.

In this study, regarding the age distribution it was seen that patients of all age group were affected but the highest numbers of bacterial corneal ulcer were found in the age group between 20-39 years (Table I). This age group was the adult people and also the earning members of the family. They lead an active outdoor life and hence their eyes are exposed to ocular trauma and infection. Similar finding were reported by Wahed MA 1981 and Musch DC 1983. But in Ghana according to Hagan Maria et al 1995the highest number of bacterial corneal ulcer patients was at age 45 years or more. Sex distribution of patients showed male and female patients of bacterial corneal ulcer were 66% and 34% in group A and 62% & 38% in group B respectively (Table-II). So the ratio between male and female patients of both groups were about 1.78:1. According to M. Srinivasan et al.1997 this ratio was 1.6:1 in South India but according to Dunlop AA et al. 1994 this ratio was 2.2:1 in Bangladesh. 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 findings of Rahman MD et al.1981, and Wahed MA 1981).

In this study the most dominated predisposing factor for developing corneal ulcer was ocular trauma which was 80.0% in group A and 76.0% in group B (Table-III). This finding correlates with the finding observed by Rahman MD 1981& Gomes DJ et al. 1983. Ocular trauma is such a condition, which creates the breach of epithelium easily and helps in invasion of bacteria leading to corneal ulcers. The other predisposing factors were dacryocystitis 3(6.0%) in group A and 2 (4.0%) in group B and no known factor was found in 7 (14.0%) in group A and 10(20%) in group B. Among the symptoms pain was the most common as complained by 100% patients of both treatment groups. Pain is due to exposure and irritation of sensory nerve endings of the cornea, iritis and increased intraocular pressure produced by hypopyon. Other symptoms of different patients were foreign body sensation, watering, purulent discharge, redness, photophobia, blurring of vision. Photophobia was due to blepharospasm set up by corneal irritation, which became greatly increased on the slightest attempt to separate the lids especially attempted in bright light. Watering was complained due to reflex secretion of tear following irritation of exposed corneal nerve endings. 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.

In the line graph showed sum of the symptoms score during baseline evaluation & 1 st to 4th follow up. Both gatifloxacin and ciprofloxacin reduces the symptoms of bacterial corneal ulcer. But reduction of symptom in patients treated with gatifloxacin is quicker and earlier than in patients treated with ciprofloxacin.

Regarding clinical signs it was evident that almost all cases were presented with congestion of conjunctiva (both conjunctival & ciliary), corneal ulcer, some with hypopyon and all corneal ulcers were fluorescein stain positive. (In table IV) There was no marked change of congestion of conjunctiva in 1st follow up. But congestion of conjunctiva was markedly reduced in group A than group B in 2 nd follow up (in group A it was severe 02(04.0%), moderate in 09(18.0%), mild in 32(64%) and absent in 07 patient (14%) in group B it was severe in 05 patient (10%), moderate in 19 patient (38%), mild in 23 patient (46%) and absent in 03 patient (6%).) With P value =0.047 which is statistically significant. In subsequent follow up congestion of conjunctiva reduces gradually in both groups. But it is not statistically significant in between groups.

In table V showed distribution of site of bacterial corneal ulcer , which is central in 50%, 44.0%, paracentral in 38.0%, 38.0% & peripheral 12.0%,18.0% in group A & B. Site of corneal ulcers in both groups were almost similar (not statistically significant). According to the study of Parmar P et al: 2006 it was central in 36%, & 40.0%, paracentral,peripheral 64%, 68% both in gatifloxacin and ciprofloxacin groups respectively .

In table VI it was observed that during baseline evaluation ulcer size was 1-2mm in 7 patients (14%) in group A and in group B 9 patients (18%), >2-3mm 20 patients (40%) in group A and in group B 21 patients (42%) & >3-4mm 14 patients (28%) in group A and in group B 13 patients (26%). No statistical significance was there. But in 2 nd follow up complete healing was found in 14 patients (28%) in group A and 5 patients (10%) in group B. Gradually the ulcer size reduced in both groups but no statistically significant difference was seen on subsequent follow up in between groups.

Table VII showed the distribution of patients with hypopyon among the corneal ulcer patients. In baseline evaluation hypopyon was present in 22 patients(44%) in group A & in 17 patients (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 =0.015.

Table VIII showed during baseline evaluation all patients were found fluorescein stain positive in both groups. During 2nd follow up it was 72% positive & 28% negative in group A, 90% positive & 10% negative in group B. It was statistically significant (P value =0.045).

(Table IX) We include only clinically diagnosed bacterial corneal ulcers & excluded which showed fungi on direct microscopy. Gram staining of corneal scraping showed gram-positive organism in 66 cases and gram-negative in 21 cases. Among gram positive cases there was growth of organism 33cases, no growth in 02 cases in group A & there was growth of organism 30 cases, no growth in 01 cases in group B, Among 21 gram negetive cases there was growth of organism 11 cases, no growth in 01 cases in group A & there was growth of organism 08 cases, no growth in 01 cases in group B. There was no statistically significant difference between two groups. Presence of organism in gram staining but on subsequent culture no growth may be due to presence of dead bacteria in corneal scraping.

In table X: Culture & sensitivity pattern of bacteria isolated from bacterial corneal ulcer.

Gatifloxacin were more sensitive (98.4%) to gram positive cocci than to ciprofloxacin (80.95%). It is statistically significant p value = 0.001. Streptococcus pneumoniae & Staphylococcus epidermidis showed more gatifloxacin sensitivity (100%) than to ciprofloxacin(74.10% & 69.20%). It is statistically significant p value = 0.025 & 0.047 respectively. Other gram positive & gram negative isolates did not showed any significance in their sensitivity pattern. This study well correlates with the study of Parmar P et al: 2006 with p value = 0.001 for gram-positive cocci and P < .001 for Streptococcus pneumoniae and P = .005 for Staphylococcus epidermidis.

Table XI: compares the in vivo clinical response of ulcers in both goups. A significantly higher proportion of ulcers in the group A exhibited complete healing compared with those in the group B (42 eyes out of 44 [95.4%] vs 29 eyes out of 38 [76.3%]; P = 0.049). This study well correlates with the study of Parmar P et al: 2006 GAT group exhibited complete healing compared with those in the CIP group (39 eyes [95.1%] vs 38 [80.9%]; P = .

042. 32 corneal ulcers patients (97%) were completely healed out of 33 patients with gatifloxacin and 21 corneal ulcers patients (70%) were completely healed out of 30 patients with ciprofloxacin (caused by gram-positive bacteria). Here p is value 0.003 in between the groups which is statistically significant. All ulcer caused by Streptococcus pneumoniae were healed by gatifloxacin but 64.3% patients caused by Streptococcus pneumoniae healed with ciprofloxacin p value among the group A & B is 0.030. This is statistically significant.

This study well correlates with the study of Parmar P et al: 2006 Staphylococcus epidermidis P = .043 and by Streptococcus pneumoniae P = .007. Clinical response of other grampositive & gram-negative bacteria of both groups showed no significant difference. This superior action of gatifloxacin as compared with ciprofloxacin had been reported by Mather R, et al. 2002; & Kaliaimurthy J, et al. 2005; Table XII: 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.

Condition of Staphylococcus aureus infected corneal ulcer was gradually deteriorating during therapy because it was not gatifloxacin sensitive but sensitive to ciprofloxacin, the ulcer was healed with ciprofloxacin. Pseudomonas aeruginosa infected corneal ulcer was also gradually deteriorating during therapy and developed perforation for which conjunctival hooding with tarsorrhaphy was done but despite all patient developed endophthalmitis. In group B 8 culture positive & 1 culture negative corneal ulcer failed to heal. There was perforation of 3 Staphylococcus epidermidis +ve cases. The ulcer of these cases were large size & 2 of them

were deep seated. They were managed with conjunctival hooding with tarsorrhaphy. 2 ulcers with Streptococcus pneumonae +ve cases developed endophthalmitis despite all measures taken. Condition of the other 4 cases were gradually deteriorating during therapy because it was not sensitive to ciprofloxacin but sensitive to gatifloxacin, the ulcer was healed with gatifloxacin. Table XIII: showed slow response in Gatifloxacin and Ciprofloxacin groups.

There was little improvement of symptoms and ulcer size was the same in 2 consecutive follow up- in group A one was Pseudomonas aeruginosa +ve corneal ulcer other is culture negative. When ulcer size was remain stationary during 2 nd follow up despite continuing treatment, tarsorrhaphy was done to help in the healing of the ulcer. Slow response in healing of the ulcer was due to inconvenience of medication by the patient. In group B 6 cases showed slow response, 2 were culture positive other 4 were culture negative. Culture +ve cases were infected by Streptococcus pneumoniae & both had chronic dacryocistitis for which DCT was on 2nd follow up as the age of those patients were 60+. Initial therapy was started with ciprofloxacin in all the cases. During 2 nd follow up as ulcer size was remain stationary so to prevent complication and to promote healing the cases were surgically treated by tarsorrhaphy, ultimately ulcers healed. (Table-XIV) Visual acuity in baseline evaluation was ranging from PL to 6/12 according to the site, size and severity of the corneal ulcer. It was almost similar in both groups. But in final follow up of the study 1 patient(2%) in group A and 2 patient(4%) in group B had no perception of light, it was due to endophthalmitis. Following haeling of corneal ulcer there was formation of corneal scar of different density. Visual acuity was improved in both groups ranging from perception of light to 6/6. Variation in the visual acuity was due to formation of corneal scar of different density.VA will be improved in further follow up when inflammatory reaction will be fully subsided. This pattern is similar to that reported from populations studied in other countries (Ormerod et al. 1986).

Chapter- 11 CONCLUSION Gatifloxacin treated corneal ulcer showed statistically clinically significance in reduction of symptoms, signs and healing of ulcer than ciprofloxacin treated cases. Complications are less in gatifloxacin group. A significantly higher percentage of ulcers that had been treated with gatifloxacin exhibited complete healing compared with those treated with ciprofloxacin. In particular, gatifloxacin had a significantly better action against gram-positive cocci both in vitro and in vivo when compared with ciprofloxacin. Because these organisms are the leading cause of keratitis worldwide, this suggests that gatifloxacin is superior to ciprofloxacin in the treatment of bacterial corneal ulcer.

Chapter- 12 LIMITATIONS OF STUDY Limitation of time Study was done from July 2008 to December 2009.

Financial limitation Government financial help was insufficient & not in time for this study.

Sample size limitation

100 patients were selected for the study. If the study was done with large sample, it will carry more accurate result.

Case selection limitation: Only 2 cases in group A and 3 cases in group B having deep ulceration, if deep ulcer was more in number the effectiveness of drug would be more definitely proved.

Chapter- 13 RECOMMENDATIONS This study should be done in different Medical College Hospitals.

Adequate financial support should be allocated by the Government for the study & at the early stage of study.

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.

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