polymerase chain reaction of intraocular fluid in cataract extraction

ASCRS 2009, San Francisco, 3 April - 8 April ASCRS 2009, San Francisco, 3 April - 8 April

Polymerase Chain Reaction Polymerase Chain Reaction of Intraocular fluid in cataract of Intraocular fluid in cataract

extractionextraction

Soon Lek Yap, M.D.Soon Lek Yap, M.D.11; Dinesh Kumar, M.D.; Dinesh Kumar, M.D.11; ; Visvaraja Subrayan, M.D.Visvaraja Subrayan, M.D.11; Sharmala Devi, PhD; Sharmala Devi, PhD22..

The authors have no financial interest in any material used in The authors have no financial interest in any material used in this studythis study

1 - Ophthalmology Department, University of Malaya1 - Ophthalmology Department, University of Malaya2 - Microbiology Department, University of Malaya2 - Microbiology Department, University of Malaya


PurposePurpose The main aim of this study is to evaluate the bacteria The main aim of this study is to evaluate the bacteria

contamination rate of the anterior chamber during cataract contamination rate of the anterior chamber during cataract extraction using real-time polymerase chain reaction (PCR) extraction using real-time polymerase chain reaction (PCR) analysis. analysis.

Post operative endophthalmitis is a potentially devastating outcome post cataract surgeryPost operative endophthalmitis is a potentially devastating outcome post cataract surgery

The incidence of endophthalmitis is 0.07-0.13%The incidence of endophthalmitis is 0.07-0.13%

Previous studies of AC contamination were based on bacteria culture, the sensitivity of 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 culture limit the detection rate of AC contamination and therefore may not reflect the actual contamination rateactual contamination rate

PCR is highly sensitive and is able to detect bacteria at a concentration of < 10PCR is highly sensitive and is able to detect bacteria at a concentration of < 1022 CFU CFU

Microscopy, although rapid, requires a relatively large concentration of bacteria (>10Microscopy, although rapid, requires a relatively large concentration of bacteria (>1044 CFU/ml)CFU/ml)


Method – Sample collectionMethod – 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


Method – Real-time PCRMethod – 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).


Real-time PCR analysisReal-time PCR analysis DNA samples were assayed in a 25 µl of preparation containing 3 µl DNA samples were assayed in a 25 µl of preparation containing 3 µl

of sample DNA and primer (10 µM) as final concentration. The 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 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, initial denaturation, 25 cycles of PCR at 94ºC, denaturation for 1 min, 60ºC of annealing for 1 min and 72ºC extension for 2 min.60ºC of annealing for 1 min and 72ºC extension for 2 min.

Real time fluorescence measurements were taken and a threshold Real time fluorescence measurements were taken and a threshold cycle (Ct) values for each sample were calculated by determining cycle (Ct) values for each sample were calculated by determining the point at which the fluorescence exceeded a threshold limit.the point at which the fluorescence exceeded a threshold limit.


Real Time PCRReal Time PCR


ResultsResults 133 pair samples were 133 pair samples were

analysed.analysed.

5 (3.8%) pre-op and 28 5 (3.8%) pre-op and 28 (21.1%) post-op samples (21.1%) post-op samples were positives.were positives.

At 6 month follow up, At 6 month follow up, none of the patients none of the patients developed developed endophthalmitisendophthalmitis


ResultsResults Further analysis did not show statistical significant Further analysis did not show statistical significant

results for patient factors such as diabetes that might results for patient factors such as diabetes that might contribute towards post-operative positive samplescontribute towards post-operative positive samples

PCR Post-op Results * DM Crosstabulation

62 43 10583.8% 72.9% 78.9%

12 16 2816.2% 27.1% 21.1%

74 59 133100.0% 100.0% 100.0%

Count% within DMCount% within DMCount% within DM

-ve

+ve

PCR Post-opResults

Total

No YesDM

Total

Χ2 test p = 0.125, >0.05; difference not significant


ResultsResults The peri-operative use of single-dose minims dilating The peri-operative use of single-dose minims dilating

eye drops did not lower the positive rate compared to eye drops did not lower the positive rate compared to multi-dose bottle eye drops.multi-dose bottle eye drops.

PCR Post-op Results * Normal Drops or Minims Crosstabulation

49 56 105

79.0% 78.9% 78.9%

13 15 28

21.0% 21.1% 21.1%

62 71 133

100.0% 100.0% 100.0%

Count% within NormalDrops or MinimsCount% within NormalDrops or MinimsCount% within NormalDrops or Minims

-ve

+ve

PCR Post-opResults

Total

Normal Drops MinimsNormal Drops or Minims

Total



The experience of The experience of surgeons and surgeons and duration of surgery duration of surgery did not affect the did not affect the results significantly.results significantly.

PCR Post-op Results * Duration of surgery (mins) Crosstabulation

23 48 26 8 105

67.6% 84.2% 78.8% 88.9% 78.9%

11 9 7 1 28

32.4% 15.8% 21.2% 11.1% 21.1%

34 57 33 9 133

100.0% 100.0% 100.0% 100.0% 100.0%

Count% within Durationof surgery (mins)Count% within Durationof surgery (mins)Count% within Durationof surgery (mins)

-ve

+ve

PCR Post-opResults

Total

<30 mins 30-45 mins 45-60 mins > 60 minsDuration of surgery (mins)

Total

PCR Post-op Results * Years Of Surgeon Experience Crosstabulation

50 15 40 105

75.8% 83.3% 81.6% 78.9%

16 3 9 28

24.2% 16.7% 18.4% 21.1%

66 18 49 133

100.0% 100.0% 100.0% 100.0%

Count% within Years OfSurgeon ExperienceCount% within Years OfSurgeon ExperienceCount% within Years OfSurgeon Experience

-ve

+ve

PCR Post-opResults

Total

< 5 years 5 to 10 years specialistYears Of Surgeon Experience

Total




DiscussionDiscussion Data of some AC contamination studies from recent years are Data of some AC contamination studies from recent years are

shown in the following tableshown in the following table

Studies from different countriesStudies from different countries Sample Sample sizesize

Contamination Contamination raterate

Leong JK et al (Australia, JCRS 2002)Leong JK et al (Australia, JCRS 2002) 9696 0%0%

Bauz M et al (Hungary, JCRS 2006)Bauz M et al (Hungary, JCRS 2006) 9797 2%2%

Feys J et al (France, JFO 1999)Feys J et al (France, JFO 1999) 10921092 5%5%

Samad A et al (Canada, AJO 1995)Samad A et al (Canada, AJO 1995) 103103 5%5%

TA CN et al (US, AJO 2004)TA CN et al (US, AJO 2004) 112112 8%8%

Ang EL et al (Malaysia, unpublished data Ang EL et al (Malaysia, unpublished data 2006)2006)

333333 1%1%


DiscussionDiscussion AC contamination rates are different in different AC contamination rates are different in different

countries and may be due to different countries and may be due to different techniques in collecting the samples and techniques in collecting the samples and different pre-op preparationdifferent pre-op preparation

In general, AC contamination rates are lower in In general, AC contamination rates are lower in recent years and may be due to the use of recent years and may be due to the use of povidone iodine eye drops prior to the operationpovidone iodine eye drops prior to the operation

Contamination rate detection by the use of PCR Contamination rate detection by the use of PCR is higheris higher


ConclusionConclusion AC contamination rate in cataract surgery is still AC contamination rate in cataract surgery is still

high, around 20% as shown in our study.high, around 20% as shown in our study.

PCR is sensitive in detecting AC contamination PCR is sensitive in detecting AC contamination although the cost is highalthough the cost is high

Better aseptic techniques, postoperative Better aseptic techniques, postoperative antibiotics and innate defense mechanisms are antibiotics and innate defense mechanisms are responsible for the very low incidence of responsible for the very low incidence of endophthalmitisendophthalmitis


ReferencesReferences1. 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.

2. 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

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

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

5. 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

polymerase chain reaction of intraocular fluid in cataract extraction

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