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

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 lt 102 CFU
• Microscopy, although rapid, requires a relatively
  large concentration of bacteria (gt104 CFU/ml)

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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

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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).

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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.

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Real Time PCR
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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

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Results

• Further analysis did not show statistical
  significant results for patient factors such as
  diabetes that might contribute towards
  post-operative positive samples

?2 test p 0.125, gt0.05 difference not
significant
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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.

?2 test p 0.982, gt0.05 difference not
significant
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• The experience of surgeons and duration of
  surgery did not affect the results significantly.

?2 test p 0.662, gt0.05 difference not
significant
?2 test p 0.251, gt0.05 difference not
significant
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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) 96 0
Bauz M et al (Hungary, JCRS 2006) 97 2
Feys J et al (France, JFO 1999) 1092 5
Samad A et al (Canada, AJO 1995) 103 5
TA CN et al (US, AJO 2004) 112 8
Ang EL et al (Malaysia, unpublished data 2006) 333 1
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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

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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

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References

1. Leong JK, Shah R, McCluskey PJ, et al. Bacterial
   contamination of the anterior chamber during
   phacoemulsification cataract surgery. J Cataract
   Refrac Surg 2002 28826-33.
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 321691-3
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 22213-4
4. Samad A, Solomon LD, Miller MA, et al. Anterior
   chamber contamination after uncomplicated
   phacoemulsification and intraocular lens
   implantation. Am J Ophthalmol 1995 120143-52
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 137662-7