Accepted Manuscript The efficacy of corneal debridement in the treatment of microsporidial keratoconjunctivitis: A prospective randomized clinical trial Sujata Das, MS, FRCS (Glasg.) Batriti S. Wallang, DO Savitri Sharma, MD Yogesh V. Bhadange, MD Praveen K. Balne, MSc Srikant K. Sahu, MS PII:

S0002-9394(14)00133-0

DOI:

10.1016/j.ajo.2014.02.050

Reference:

AJOPHT 8855

To appear in:

American Journal of Ophthalmology

Received Date: 6 October 2013 Revised Date:

25 February 2014

Accepted Date: 25 February 2014

Please cite this article as: Das S, Wallang BS, Sharma S, Bhadange YV, Balne PK, Sahu SK, The efficacy of corneal debridement in the treatment of microsporidial keratoconjunctivitis: A prospective randomized clinical trial, American Journal of Ophthalmology (2014), doi: 10.1016/j.ajo.2014.02.050. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The efficacy of corneal debridement in the treatment of microsporidial keratoconjunctivitis: A prospective randomized clinical trial Sujata Das, MS, FRCS (Glasg.)

1

Batriti S Wallang, DO

2

Savitri Sharma, MD

1

Yogesh V Bhadange, MD

2

Praveen K Balne, MSc

1

Srikant K Sahu, MS

1

Cornea and Anterior Segment Service

2

Ocular Microbiology Service

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L. V. Prasad Eye Institute, Bhubaneswar, Odisha, India, 751024

: Microsporidia, Keratoconjunctivitis, Debridement

Financial Support

: None.

Conflict of Interest

: No conflicting relationship exists for any author. : Debridement for treatment of microsporidial keratoconjunctivitis.

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Running Head

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Key Word

Address for Correspondence Sujata Das, MS, FRCS (Glasg.) Consultant, Cornea and Anterior Segment Service L. V. Prasad Eye Institute Bhubaneswar, Odisha India, 751024 T: +91 (674) 398-7999 F: +91 (674) 398-7130 [email protected] Page 1 of 10

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ABSTRACT PURPOSE: To evaluate the efficacy of corneal debridement in the treatment of clinically diagnosed cases of microsporidial keratoconjunctivitis.

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DESIGN: Prospective, double-masked, randomized, clinical trial. METHODS:

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Patients with clinical features, such as multifocal, coarse, raised, punctate, round to oval

epithelial lesions in the cornea in slit-lamp examination with mild to moderate conjunctival

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congestion, suggestive of microsporidial superficial keratoconjunctivitis, were included in the prospective study. All patients were randomized into two groups. Group-1 patients underwent debridement with the help of a sterile #15 blade on a Bard-Parker handle, whereas only conjunctival swabs were taken from Group-2 patients. All patients were treated with ocular lubricants.

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RESULTS:

One hundred and twenty patients with clinical features suggestive of microsporidial superficial keratoconjunctivitis were included in the study. The mean age was 34.3±13.6 (Group-1) and

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35.8±16.2 (Group-2) years (p=0.59). The mean duration of symptoms was 6.8±3.9 (Group-1) and 7.2±4.6 (Group-2) days (p=0.61). Baseline characteristics showed no difference between

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the two groups. The primary outcome was the time taken from the presentation to complete resolution (i.e., absence of corneal lesions) of the clinical signs and symptoms. The secondary outcomes were: final visual acuity and residual corneal side-effects and/or scarring, if any. The mean resolution time of the corneal lesions was 5.7±4.0 (Group-1) and 5.9±3.9 (Group-2) days (p=0.83). There was no significant difference in final visual outcome in the two groups. No serious side effects were observed. CONCLUSION: Debridement does not have any significant advantage in terms of resolution of the corneal lesions and final visual outcome in cases of microsporial keratoconjunctivitis. Page 1 of 1

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INTRODUCTION Earlier considered a disease of immunocompromised patients, microsporidial keratoconjunctivitis has come to be known as a common ocular infection in large number of immunocompetent patients.1-6 The patients usually present with complaints of redness,

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photophobia, irritation, watering, and defective vision. It is diagnosed by clinical features and microbiological investigations.6 A number of clinical and in-vitro studies have demonstrated variable responses of microsporidia to various therapeutic agents. However, their efficacy still remains controversial.

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More recent studies and clinical observations at this Institute have suggested that

microsporidial keratoconjunctivitis is a self-limiting infection that does not require any specific

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chemotherapeutic intervention.7 Surgical intervention in the form of diagnostic and therapeutic debridement may help in faster clinical resolution by decreasing the microbial load in the corneal epithelium.8 However, there is no evidence that debridement alone helps in early resolution. It is also believed that debridement may cause stromal penetration of the organism and increase the risk of secondary infection although there are no reports of stromal keratitis

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following debridement of lesions. The present study was designed to evaluate the efficacy of corneal debridement in the treatment of keratoconjunctivitis caused by microsporidia and also to determine its side effects, if any.

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PATIENTS AND METHODS

This single-centre, double-masked, randomized clinical trial examined 120 patients. The

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prospective study has been approved by the Institution Review Board, L. V. Prasad Eye Institute, Hyderabad Eye Research Foundation, and registered with the Clinical Trials Registry (India) (No. CTRI/2011/11/ 002132). It was conducted at the L. V. Prasad Eye Institute, Bhubaneswar, India. Informed written consent was obtained from each participant before inclusion in the study. Patients were prospectively recruited between July 2011 and August 2012.

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INCLUSION AND EXCLUSION CRITERIA Patients presenting with clinically diagnosed superficial epithelial microsporidial keratitis or keratoconjunctivitis having a minimum of 10 discrete epithelial or subepithelial lesions at presentation were included in the study. Informed consent was taken from all patients included

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in the study. The exclusion criteria included clinically diagnosed cases of superficial microsporidial keratoconjunctivitis with less than 10 discrete lesions and/or presence of stromal infiltrates at presentation.

Patients who adhered to a follow-up visit schedule either on day 5±2 days or resolved publication,7 where resolution time was 5±2 days.

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SAMPLE SIZE, RANDOMIZATION AND MASKING

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before this day were included in the analysis. We decided this criterion based on our previous

A computer-generated randomization number was assigned to each patient at the time of initiation of treatment. As this was a double-blinded (double-masked) study, both the study participants and the evaluating ophthalmologists were masked to the information regarding the participants undergoing debridement/conjunctival swab or otherwise. The evaluating

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ophthalmologists were not informed about the assigned treatment group of the patient. Another ophthalmologist who was not involved in the evaluation process during follow-up visits of the patients did debridement or took conjunctival swab. Conjunctival swab was taken to mask the

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patient.

A sample size of 36 in each group was considered sufficient to detect a difference of at

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least 2 days between the means of the two study groups and the standard deviations of 3 days in each of the two study groups with 80% power and 5% level of significance.

PRIMARY AND SECONDARY OUTCOME MEASURES The primary outcome was the time taken from the presentation to complete resolution of the clinical signs and symptoms. Resolution was defined as absence of corneal lesions. The secondary outcomes were: (1) final visual acuity, and (2) residual corneal side-effects and/or scarring, if any.

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PROCEDURES At the baseline (i.e. day 0), demographic information and detailed medical history was obtained from each patient. Visual acuity was measured using a Snellen chart, and slit-lamp examination was performed on all patients. All patients who exhibited clinical features of microsporidial

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keratoconjunctivitis, such as diffuse, multifocal, coarse, raised, punctate, round to oval epithelial lesions in the cornea in slit-lamp examination were randomized into two groups.

Debridement of all the lesions was done with the help of a sterile no. 15 blade on a BardParker handle (Sharp Edge Industries, Ahmedabad, India) for patients in the debridement

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group after instillation of topical anaesthetic (0.5% proparacaine hydrochloride) eye drop. The scraping was smeared on 2 glass slides and was subjected to direct smear examination after being stained with 10% potassium hydroxide with 0.1% calcofluor white (fluorescence

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microscopy) and Gram or Giemsa or modified Ziehl-Neelsen stain (1% H2SO4). Microscopic slides were examined for all patients except one (inadequate sample). The corneal scraping was also inoculated onto 1 culture medium (chocolate agar/blood agar) which was incubated at 37OC for 2 weeks. Sample for PCR was collected from all patients. After sending corneal scraping for microbiological inverstigation, remaining epithelial lesions and surrounding

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epithelium were removed with help of no. 15 blade. In general, epithelium surrounding the corneal lesions is relatively loose. Conjunctival sample was taken from the lower fornix with the help of a rayon swab after instillation of topical anaesthetic (0.5% proparacaine hydrochloride)

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eye drop for patients in the non-debridement group. Corneal scrapings and conjunctival swabs were subjected to polymerase chain reaction (PCR) for microsporidia. PCR analysis was done

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using a previously described small subunit rRNA based panmicrosporidian PCR that would amplify all medically important species of microsporidia.9 All patients were prescribed with artificial tear substitute, 4-times a day, and were examined on day 0, 3±1, 7±1 and at weekly intervals until resolution of clinical signs and symptoms. Assessment of patients on follow-up visits was done by an ophthalmologist masked to the treatment.

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STATISTICAL ANALYSIS Baseline characteristics were compared using unpaired t test. The chi-square test was used to compare proportions. To compare the time for resolution and intergroup visual acuity in both the groups, unpaired t test was used.

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RESULTS PATIENT DISPOSITION AND DEMOGRAPHICS

One hundred and twenty eyes of 120 patients (debridement group, 58; non-debridement group,

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62) were recruited. Baseline characteristics (age, gender, duration of symptoms, clinical

features) showed no relevant difference between the two groups (Table-1). Flowchart detailing

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disposition of patients with microsporidial keratoconjunctivitis who were included in the clinical trial is shown in Figure-1. Patients who had adhered to the scheduled follow-up visits and had corneal lesions resolved were included in the outcome analysis (i.e. resolution time, scar, final visual acuity) (debridement group, 35; non-debridement group, 35). None of the study patients were immunocompromised in either group.

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MICROBIOLOGICAL RESULT

Microsporidial spores were demonstrated in direct microscopic examination by one or more staining methods in 54 out of 57 (94.7%) corneal scrapings from the debridement group. In this group the microsporidial DNA was detected in corneal scrapings of 54 out of 58 (93.1%) cases.

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On the other hand, PCR for microsporidial DNA was positive from conjunctival swab in 43/62

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(69.3%) cases in the non-debridement group.

EFFICACY OF DEBRIDEMENT RESOLUTION OF CORNEAL LESIONS

We defined resolution time as the time required for the corneal epithelial infiltrates to disappear completely (no infiltrates visible on slit-lamp examination). The mean time taken to resolve was 5.7±4.0 days and 5.9±3.9 days in the debridement and non-debridement groups, respectively (p=0.83) (Table-2). 67.5% (27/40) and 64.3% (27/42) cases resolved within 1 week of presentation in the debridement and non-debridement groups, respectively (p=0.94) (Figure-1) Page 5 of 10

ACCEPTED MANUSCRIPT VISUAL ACUITY

The visual acuity was measured using the Snellen chart. Logarithmic conversion was carried out to compare the visual acuity before treatment with that after treatment in both groups. The mean visual acuity of the patients at the time of presentation was 0.24±0.32 and 0.24±0.30 in the debridement and non-debridement groups, respectively (p=1.00) (Table-2). The final visual

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acuity after treatment in both the groups was 0.14±0.25 and 0.18±0.32 in the debridement and non-debridement groups, respectively (p=0.56) (Table-2). There was no significant difference between the groups in the extent of improvement.

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FINAL CLINICAL OUTCOME

Final outcome was analyzed on the basis of percentage of clear cornea or scar (nummular or

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diffuse) in either group at the last visit. Residual scar, if existed, was superficial in all cases without affecting the visual acuity. While 34.3% (12/35) patients had scar in the debridement group, 28.6 % (10/35) had scar in the non-debridement group (p=0.80) (Table-2). SAFETY OF DEBRIDEMENT

Significant worsening was defined as subjective increase in any one of the symptoms (i.e.

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redness, pain, tearing), and/or increase of any of the ocular signs (i.e, eye lid swelling, conjunctival congestion), and significant increase in the number of lesion (>10). None of our

DISCUSSION

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study patients had worsened.

Keratoconjunctivitis is the most common clinical manifestation of ocular microsporidiosis.

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Though microsporidial keratoconjunctivitis has come to be known as a common ocular infection, there has been no consensus on the definitive management. Various drug treatments have been advocated including the use of albendazole, itraconazole, metronidazole, propamidine isethionate, benzimidazole, fumagilin, voriconazole, polyhexamethylene biguanide and chlorhexidine, either alone or in combination.2,3,10-16 We conducted a randomized clinical trial to evaluate the efficacy of 0.02% polyhexamethylene biguanide (PHMB) in the treatment of superficial keratoconjunctivitis and concluded that it did not offer any significant advantage over placebo.7 There was no significant difference in resolution time, cure time and final visual outcome between the two groups. We concluded self-limiting nature of the disease. However, Page 6 of 10

ACCEPTED MANUSCRIPT we did not exclude the role of debridement in healing of keratoconjunctivitis. Debridement may help in faster clinical resolution by decreasing the microbial load in the corneal epithelium. However, there is no evidence that debridement alone helps in early resolution. Fan et al had reported that repeated corneal swabbing with help of cotton swabs could eradicate corneal lesions in approximately one week.17

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In this study we included all cases of clinically diagnosed microsporidial keratoconjunctivitis. Most corneal lesions in the patients were superficial and distributed diffusely, thus enabling corneal scraping to work like debridement thereby reducing the load of organisms in cases of

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superficial keratoconjunctivtis. Confirmed by microscopic demonstration of microsporidial

spores in corneal scrapings, we have shown earlier that a reliable clinical diagnosis can be made in over 89% cases of keratoconjunctivtis.6 In this series, microscopic examination was

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positive in 94.7% cases. Expectedly, PCR positivity from conjunctival swab was less in comparison to corneal scraping, probably due to lower load of organisms in conjunctiva compared to corneal lesions. We have also observed that the epithelium is loose in these cases, thereby making removal of lesions possible by gentle scraping with the help of a sterile no. 15 blade on a Bard-Parker handle.6

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Although, there is no significant difference in the resolution time between the two groups, it is marginally higher in the non-debridement group. Therefore, we recommend therapeutic debridement of the active lesions when the patient is symptomatic and/or worsening and the

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lesions are increasing with time. However, we believe that despite marginal worsening, the lesions would resolve with time. Although a few patients in both groups were more symptomatic

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in the second visit, none required exiting from the study. The mean resolution time with repeated corneal swabbing was found to be 6.6 days in an earlier study, which is similar to our finding. 17 While debridement is one time procedure, corneal swabbing has to be repeated multiple times.17

Visual outcome after resolution of microsporidial keratitis often was satisfactory. Residual subepithelial scars usually did not affect visual acuity. There was no significant difference in final visual acuity between both the groups. Contrary to the common belief that debridement may cause stromal penetration of the organism; we did not come across any stromal keratitis following debridement of lesions. Page 7 of 10

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ACKNOWLEDGMENTS / DISCLOSURE (a) Funding/Support

: The study was supported by the “Hyderabad Eye Research

Foundation, Hyderabad, India”. (b) Financial Disclosures

: There is “No Financial Conflicts of Interest” among the

(c) Contributions to Authors

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authors. : Involved in:

a. Design of Study (S.D.),

S.K.S.),

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b. Conduct of Study (S.D., B.S.W., S.S., Y.V.B., P.K.B.,

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c. Collection of Data (B.S.W., Y.V.B., P.K.B.), d. Management, Analysis, and Interpretation of Data (S.D., S.S.),

e. Preparation of the Manuscript (S.D., B.S.W.), and

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f. Review and Approval of the Manuscript (S.S.). (d) Other Acknowledgements : None

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REFERENCES

1. Chan CM, Theng JT, Li L, Tan DT. Microsporidial keratoconjunctivitis in healthy individuals:

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a case series. Ophthalmology 2003; 110(7): 1420-1425. 2. Sridhar MS, Sharma S. Microsporidial keratoconjunctivitis in a HIV-seronegative patient treated with debridement and oral itraconazole. Am J Ophthalmol 2003; 136(4):745-746. 3. Joseph J, Sridhar MS, Murthy S, Sharma S. Clinical and microbiological profile of microsporidial keratoconjunctivitis in southern India. Ophthalmology 2006; 113(4):531-537. 4. Das S, Sharma S, Sahu SK, Nayak SS, Kar S. New microbial spectrum of epidemic keratoconjunctivitis: clinical and laboratory aspects of an outbreak. Br J Ophthalmol 2008; 92(6): 861-862. Page 8 of 10

ACCEPTED MANUSCRIPT 5. Loh RS, Chan CM, Ti SE, Lim L, Chan KS, Tan DT. Emerging prevalence of microsporidial keratitis in Singapore: epidemiology, clinical features, and management. Ophthalmology 2009; 116(12): 2348-2353. 6. Das S, Sharma S, Sahu SK, Nayak SS, Kar S. Diagnosis, clinical features and treatment

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outcome of microsporidial keratoconjunctivitis. Br J Ophthalmol 2012; 96(6): 793-795. 7. Das S, Sahu SK, Sharma S, Nayak SS, Kar S. Clinical trial of 0.02% polyhexamethylene biguanide versus placebo in the treatment of microsporidial keratoconjunctivitis. Am J Ophthalmol 2010; 150(1): 110-115.

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8. Sridhar MS, Sharma S. Microsporidial keratoconjunctivitis in a HIV-seronegative patient treated with debridement and oral itraconazole. Am J Ophthalmol 2003; 136(4): 745-746.

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9. Reddy AK, Balne PK, Gaje K, Garg P. PCR for the diagnosis and species identification of microsporidia in patients with keratitis. Clin Microbiol Infect 2011; 17(3): 476-478. 10. Metcalfe TW, Doran RM, Rowlands PL, Curry A, Lacey CJ. Microsporidial keratoconjunctivitis in a patient with AIDS. Br J Ophthalmol 1992; 76(3): 177-178. 11. Diesenhouse MC, Wilson LA, Corrent GF, Visvesvara GS, Grossniklaus HE, Bryan RT.

1993; 115(3): 293-298.

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Treatment of microsporidial keratoconjunctivitis with topical fumagillin. Am J Ophthalmol.

12. Gritz DC, Holsclaw DS, Neger RE, Whitcher JP Jr, Margolis TP. Ocular and sinus

241-243.

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microsporidial infection cured with systemic albendazole. Am J Ophthalmol 1997; 124(2):

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13. Rosberger DF, Serdarevic ON, Erlandson RA, Bryan RT, Schwartz DA, Visvesvara GS, Keenan PC. Successful treatment of microsporidial keratoconjunctivitis with topical fumagillin in a patient with AIDS. Cornea 1993; 12(3): 261-265. 14. Rossi P, Urbani C, Donelli G, Pozio E. Resolution of microsporidial sinusitis and keratoconjunctivitis by itraconazole treatment. Am J Ophthalmol 1999; 127(2): 210-212. 15. Yee RW, Tio FO, Martinez JA, Held KS, Shadduck JA, Didier ES. Resolution of microsporidial epithelial keratopathy in a patient with AIDS. Ophthalmology 1991; 98(2): 196-201. Page 9 of 10

ACCEPTED MANUSCRIPT 16. Khandelwal SS, Woodward MA, Hall T, Grossniklaus HE, Stulting RD. Treatment of microsporidia keratitis with topical voriconazole monotherapy. Arch Ophthalmol 2011; 129(4): 509-510.

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17. Fan NW, Lin PY, Chen TL, Chen CP, Lee SM. Treatment of microsporidial keratoconjunctivitis with repeated corneal swabbing. Am J Ophthalmol 2012; 154(6): 927933.

FIGURE LEGEND FIGURE-1

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Flowchart detailing disposition of patients with microsporidial keratoconjunctivitis who were included in the clinical trial.

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ACCEPTED MANUSCRIPT Table-1:

Comparison of Baseline Characteristics of Patients with Microsporidial Keratoconjunctivitis Who Enrolled in the Study Debridement Group (n=58)

Non-debridement Group (n=62)

Age (years)

p Value 0.59

35.8 (16.2)

Range

16 to 69

8 to 77

95% CI

30.7 to 37.9

31.7 to 39.9

Gender (male-to-female)

51:7

45:17

0.06

Eye (right-to-left)

28:30

29:33

0.98

Duration of symptoms Mean (SD)

6.8 (3.9) 2 to 15

95% CI Number of corneal lesions (>20)

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0.61

7.2 (4.6) 2 to 30

5.8 to 7.8

6.9 to 8.4

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43

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CI = confidence interval; SD = standard deviation

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Range

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34.3 (13.6)

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Mean (SD)

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1.00 58 0.24 (0.32) 0 to 2 0.16 to 0.32 35 5.7 (4.0) 1 to 17 4.3 to 7.1

Scar

12 (of 35)

62 0.24 (0.30) 0 to 1.3 0.16 to 0.32

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35 5.9 (3.9) 2 to 22 4.6 to 7.2

10 (of 35)

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Resolution time (days) No. Mean (SD) Range 95% CI Final VA (logMAR) No.

35

0.14 (0.25)

0.18 (0.32)

Range

0 to 1.3

0 to 1.3

95% CI

0.05 to 0.23

0.07 to 0.29

Mean (SD)

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CI = confidence interval; SD = standard deviation; logMAR = logarithm of the minimal angle of resolution; VA = visual acuity.

0.80 0.56

35

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p Value

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VA at presentation (logMAR) No. Mean (SD) Range 95% CI

Non-debridement Group

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Table-2:

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This prospective, double-masked, randomized, clinical trial was conducted to evaluate the efficacy of corneal debridement in the treatment of clinically diagnosed cases of microsporidial keratoconjunctivitis. Therapeutic debridement of corneal lesions does not have any significant

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advantage in terms of resolution of the corneal lesions and final visual outcome in cases of

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microsporial keratoconjunctivitis.