Transcript 191: Detection of P Aeruginosa in Contact Lens

Detection of P Aeruginosa in Contact
Lens–Related Infectious Keratitis Using
Polymerase Chain Reaction
Soon Lek Yap1, M.D.; Visvaraja Subrayan1 M.D.;
Nadir Ali Mohamed Ali1 M.D.; Shamala Devi2 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
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
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.
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
Amplicon
Size(bp)
P.
aeruginosa
lasA
Forward:
5’-AGTTGTCGCGGCGCTACTAC-3’
Reverse:
5’-GCTCACCTGGATCTGGTCCA-3’
125 bp
Eubacteria
16SrR
NA
Forward:
5’- CCTAACACATGCAAGTCGA –3’
Reverse:
5’- CCTCTCAGACCAGTTA- 3’
World Cornea Congress, Boston, 7-9 April 2010
225 bp
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)
World Cornea Congress, Boston, 7-9 April 2010
Results
Culture
PCR
Total
Total
-ve
+ve
-ve
4
1
5
+ve
1
4
5
5
5
10
World Cornea Congress, Boston, 7-9 April 2010
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 culturepositive 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.
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.
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.
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;36:34926.
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.
World Cornea Congress, Boston, 7-9 April 2010