ASCRS 2009, San Francisco, 3 April - 8 April 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, PhD 2. 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 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 %  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 < 10 2 CFU  Microscopy, although rapid, requires a relatively large concentration of bacteria (>10 4 CFU/ml)

ASCRS 2009, San Francisco, 3 April - 8 April 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 ml AC fluid were collected from the initial side port   Post-operative samples of 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 Method – Real-time PCR   DNA extraction was performed by heating the samples to 95 o C for 5 mins and stored in -20 o C   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 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 Real Time PCR

ASCRS 2009, San Francisco, 3 April - 8 April 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 Results  Further analysis did not show statistical significant results for patient factors such as diabetes that might contribute towards post-operative positive samples Χ 2 test p = 0.125, >0.05; difference not significant

ASCRS 2009, San Francisco, 3 April - 8 April 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. Χ 2 test p = 0.982, >0.05; difference not significant

ASCRS 2009, San Francisco, 3 April - 8 April  The experience of surgeons and duration of surgery did not affect the results significantly. Χ 2 test p = 0.662, >0.05; difference not significant Χ 2 test p = 0.251, >0.05; difference not significant

ASCRS 2009, San Francisco, 3 April - 8 April Discussion  Data of some AC contamination studies from recent years are shown in the following table Studies from different countries Sample size Contamination rate Leong JK et al (Australia, JCRS 2002) 960% Bauz M et al (Hungary, JCRS 2006) 972% Feys J et al (France, JFO 1999) 10925% Samad A et al (Canada, AJO 1995) 1035% TA CN et al (US, AJO 2004) 1128% Ang EL et al (Malaysia, unpublished data 2006) 3331%

ASCRS 2009, San Francisco, 3 April - 8 April 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 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 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: 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: 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: Samad A, Solomon LD, Miller MA, et al. Anterior chamber contamination after uncomplicated phacoemulsification and intraocular lens implantation. Am J Ophthalmol 1995; 120: 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