PCR-based evidence of bacterial involvement in eyes with suspected intraocular infection.
OBJECTIVE
To assess the usefulness of polymerase chain reaction (PCR) in detection of bacteria in ocular samples.


METHODS
Thirty-seven samples (aqueous and vitreous) were collected from 25 eyes showing typical symptoms and clinical signs of bacterial endophthalmitis. Ocular samples were also collected from 38 eyes that underwent routine surgery and from 15 eyes with intraocular inflammation due to nonbacterial causes. Panbacterial PCR was performed with a nested pair of 16S rRNA gene primers. Subsequent bacterial identification was completed for 18 paired samples (nine eyes) using restriction fragment length polymorphism (RFLP) and DNA sequencing.


RESULTS
A 100% concordance was obtained between PCR and culture-positive samples. A PCR product was amplified from all 37 intraocular samples from eyes with suspected infection, whereas only 15 of 22 vitreous samples and 5 of 15 aqueous samples were culture positive. Culture-negative PCR-positive samples contained a preponderance of gram-negative bacterial sequences. Cloning and DNA analysis revealed 30 DNA sequences and included eight bacterial 16S rDNA, which currently remain unidentifiable. The presence of bacterial DNA was associated with an inflammatory response suggestive of infection and not colonization. All 15 samples from inflamed eyes with diverse uveitis diagnoses were PCR negative. The false-positive rate, due to contamination during sampling, was 5%.


CONCLUSIONS
Bacterial DNA was detected in all patients with typical clinical signs of endophthalmitis. Gram-negative organisms seem to play a much more important role in the pathogenesis of this disease than previously thought. PCR-based techniques have great value in the confirmation of the diagnosis of bacterial endophthalmitis especially in culture-negative eyes.
Okhravi
N
N
Departments of Clinical Ophthalmology, The Institute of Ophthalmology and University College London, United Kingdom. nokhravi@hgmp.mrc.ac.uk
Adamson
P
P
Carroll
N
N
Dunlop
A
A
Matheson
M M
MM
Towler
H M
HM
Lightman
S
S
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001082:Aqueous Humor / Q000382:microbiology; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis*; D004275:DNA, Ribosomal / Q000235:genetics; D009877:Endophthalmitis / Q000175:diagnosis / Q000382:microbiology*; D015818:Eye Infections, Bacterial / Q000175:diagnosis / Q000382:microbiology*; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D011237:Predictive Value of Tests; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D014605:Uveitis / Q000175:diagnosis / Q000382:microbiology; D014822:Vitreous Body / Q000382:microbiology
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S