Polymerase Chain Reaction of Intraocular fluid in cataract extraction

1. Polymerase Chain Reaction of Intraocular fluid in cataract extraction Soon Lek Yap, M.D.1; Dinesh Kumar, M.D.1; Visvaraja Subrayan, M.D.1; Sharmala Devi, PhD2. 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 ASCRS 2009, San Francisco, 3 April - 8 April

2. Purpose • The main aim of this study is to evaluate the bacteria contamination rate of the anterior chamber during cataract extraction using real-time polymerase chain reaction (PCR) analysis. • Post operative endophthalmitis is a potentially devastating outcome post cataract surgery • The incidence of endophthalmitis is 0.07-0.13% • Previous studies of AC contamination were based on bacteria culture, the sensitivity of culture limit the detection rate of AC contamination and therefore may not reflect the actual contamination rate • PCR is highly sensitive and is able to detect bacteria at a concentration of < 102 CFU • Microscopy, although rapid, requires a relatively large concentration of bacteria (>104 CFU/ml) ASCRS 2009, San Francisco, 3 April - 8 April

3. Method – Sample collection • All patients received pre-operative mydriatics, local anaesthetics and 2 drops of 5% povidone iodine 5 minutes before the operation • None of the patients received pre-op antibiotics • Pre-operative samples of 0.05-0.10ml AC fluid were collected from the initial side port • Post-operative samples of 0.05-0.10 ml were collected at the end of the surgery prior to the injection of intracameral antibiotics or subconjunctival antibiotics • Patients with local or systemic infections, trauma cases, cases with intra-operative complications and inadequate sampling were excluded ASCRS 2009, San Francisco, 3 April - 8 April

4. Method – Real-time PCR • DNA extraction was performed by heating the samples to 95oC for 5 mins and stored in -20oC • Samples were analysed using real time PCR • The primer pairs used are specific for conserved DNA sequences encoding the 16S rRNA gene • The PCR reaction was carried out using one step SYBR Green Master mix. The assay was performed in an iC Real-Time PCR machine (BioRad, Hercules, California, USA). ASCRS 2009, San Francisco, 3 April - 8 April

5. Real-time PCR analysis DNA samples were assayed in a 25 µl of preparation containing 3 µl of sample DNA and primer (10 µM) as final concentration. The thermal cycling profile of the assay consisted of a 94ºC for 1 min for initial denaturation, 25 cycles of PCR at 94ºC, denaturation for 1 min, 60ºC of annealing for 1 min and 72ºC extension for 2 min. Real time fluorescence measurements were taken and a threshold cycle (Ct) values for each sample were calculated by determining the point at which the fluorescence exceeded a threshold limit. ASCRS 2009, San Francisco, 3 April - 8 April

6. Real Time PCR ASCRS 2009, San Francisco, 3 April - 8 April

7. Results • 133 pair samples were analysed. • 5 (3.8%) pre-op and 28 (21.1%) post-op samples were positives. • At 6 month follow up, none of the patients developed endophthalmitis ASCRS 2009, San Francisco, 3 April - 8 April

8. Results • Further analysis did not show statistical significant results for patient factors such as diabetes that might contribute towards post-operative positive samples Χ2 test p = 0.125, >0.05; difference not significant ASCRS 2009, San Francisco, 3 April - 8 April

9. Results • The peri-operative use of single-dose minims dilating eye drops did not lower the positive rate compared to multi-dose bottle eye drops. Χ2 test p = 0.982, >0.05; difference not significant ASCRS 2009, San Francisco, 3 April - 8 April

10. The experience of surgeons and duration of surgery did not affect the results significantly. Χ2 test p = 0.662, >0.05; difference not significant Χ2 test p = 0.251, >0.05; difference not significant ASCRS 2009, San Francisco, 3 April - 8 April

11. Discussion • Data of some AC contamination studies from recent years are shown in the following table ASCRS 2009, San Francisco, 3 April - 8 April

12. Discussion • AC contamination rates are different in different countries and may be due to different techniques in collecting the samples and different pre-op preparation • In general, AC contamination rates are lower in recent years and may be due to the use of povidone iodine eye drops prior to the operation • Contamination rate detection by the use of PCR is higher ASCRS 2009, San Francisco, 3 April - 8 April

13. Conclusion • AC contamination rate in cataract surgery is still high, around 20% as shown in our study. • PCR is sensitive in detecting AC contamination although the cost is high • Better aseptic techniques, postoperative antibiotics and innate defense mechanisms are responsible for the very low incidence of endophthalmitis ASCRS 2009, San Francisco, 3 April - 8 April

14. References • Leong JK, Shah R, McCluskey PJ, et al. Bacterial contamination of the anterior chamber during phacoemulsification cataract surgery. J Cataract Refrac Surg 2002; 28:826-33. • Bausz M, Fodor E, Resh MD, et al. Bacterial contamination in the anterior chamber after povidone-iodine application and the effect of lens implantation device. J Cataract Refrac Surg 2006; 32:1691-3 • Feys J, Emond JP, Meziane D, et al. Intraocular contamination during cataract surgery according to surgical technique and type of implant. J Fr Ophthalmol 1999; 22:213-4 • Samad A, Solomon LD, Miller MA, et al. Anterior chamber contamination after uncomplicated phacoemulsification and intraocular lens implantation. Am J Ophthalmol 1995; 120:143-52 • Ta CN, Egbert PR, Singh K, et al. The challenge of determining aqueous contamination rate in anterior segment intraocular surgery. Am J Ophthalmol 2004; 137:662-7 ASCRS 2009, San Francisco, 3 April - 8 April