1. World Cornea Congress, Boston, 7-9 April 2010 Detection of P Aeruginosa in Contact Lens–Related Infectious Keratitis Using Polymerase Chain Reaction Soon Lek Yap 1, M.D.; Visvaraja Subrayan 1 M.D.; Nadir Ali Mohamed Ali 1 M.D.; Shamala Devi 2 PhD Nadir Ali Mohamed Ali 1 M.D.; Shamala Devi 2 PhD The authors have no financial interest in any material used in this study 1 - Ophthalmology Department, University of Malaya 2 - Microbiology Department, University of Malaya

2. World Cornea Congress, Boston, 7-9 April 2010 Purpose ► The main aim of this study is to compare between Real Time Polymerase Chain Reaction (PCR) and conventional bacterial culture methods in the detection of pseudomonas aeruginosa in corneal ulcers

3. World Cornea Congress, Boston, 7-9 April 2010 Method ► The study duration was 6 months. ► All patients admitted with contact lens related corneal ulcer to the eye ward in our center during the study period were recruited. ► Samples from corneal scrappings were simultaneously sent at the time of admission for PCR and culture testing. ► The results of PCR and culture were compared using Mcnemar χ2-square test.

4. World Cornea Congress, Boston, 7-9 April 2010 Realtime PCR ► The eubacteria primer targeting at the 16srRNA and an in-house developed primer targeting at the Ps. aeruginosa LasR gene were used. Bacterial Strains Targe t Gene Primer set AmpliconSize(bp) P. aeruginosa lasAForward:5’-AGTTGTCGCGGCGCTACTAC-3’Reverse: 5’-GCTCACCTGGATCTGGTCCA-3’ 5’-GCTCACCTGGATCTGGTCCA-3’ 125 bp Eubacteria 16SrR NA Forward: 5’- CCTAACACATGCAAGTCGA –3’ Reverse: 5’- CCTCTCAGACCAGTTA- 3’ 225 bp

5. World Cornea Congress, Boston, 7-9 April 2010 Results ► Ten patients were recruited. ► The mean age was 31 years (20 – 45 years). ► All the patients recruited had contact lens related keratitis, of which six (60%) were found positive for Pseudomonas aeruginosa either by PCR or culture or both. ► The concordance was 66%, where 4 out 6 patients were both PCR and culture positive. ► There was no significant difference between PCR and culture in detecting Pseudomonas aeruginosa (p<0.05)

6. World Cornea Congress, Boston, 7-9 April 2010 Results CultureTotal -ve+ve PCR-ve415 +ve145 Total5510

7. Discussion ► Several studies were done using PCR for bacteria and fungal keratitis. ► In a recent large scale study involving 108 patients, Kim et al showed that PCR have a high yield of 76% in culture- positive bacteria keratitis and 94% in culture positive fungal keratitis. ► Knox et al showed similar results of 80% PCR yield in 10 culture-positive bacteria microbial keratitis. ► Rudolph et al also demonstrated the usefulness of PCR in bacterial keratitis in their small case series involving 4 patients.

8. World Cornea Congress, Boston, 7-9 April 2010 Discussion ► False positive is a major concern with the use of PCR as minute contaminant DNA from the commensal and the environment can be amplified especially with the use of eubacteria 16S rRNA primer. ► Meticulous aseptic technique in sample collection, handling and processing can reduce the contamination rates. ► The use of primers specific for Pseudomonas aeruginosa lasA gene can also increase the specificity of the assay and reduce the contamination and false positive rates. ► All the samples positive for the eubacteria primer were positive for the pseudomonas aeroginosa lasA gene in this study.

9. World Cornea Congress, Boston, 7-9 April 2010 Conclusion ► This study showed that PCR is, at least, as good as conventional cultures in detecting Pseudomonas aeruginosa. ► PCR has the advantage of a rapid result as compared to culture which provide us with a valuable guide for the choice of antibiotics in the early treatment of cornea ulcer.

10. World Cornea Congress, Boston, 7-9 April 2010 References 1. Stehling EG, Silveira WD, Leite Dda S. Study of biological characteristics of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis and from patients with extra-pulmonary infections. Braz J Infect Dis. 2008;12(1):86-8. 2. Kim E, Chidambaram JD, Srinivasan M, Lalitha P, Wee D, Liteman TM, Whitcher JP, Gelder RNV. Prospective comparison of microbial culture and polymerase chain reaction in the diagnosis of corneal ulcer. Am J Ophthalmo 2008;146:714-23. 3. Knox CM, Cevellos V, Dean D. 16S ribosomal DNA typing for identification fo pathogens in patients with bacterial keratitis. J Clin Microbiol 1998;36:3492- 6. 4. Rudolph T, Welinder-Olsson C, Lind-Brandberg L, Stenevi U. 16S rDNA PCR analysis of infectious keratitis: a case series. Acto Ophthalmol Scand 2004;82:463-7.