PCR-based evidence of bacterial involvement in eyes with suspected intraocular infection

PURPOSE. TO assess the usefulness of polymerase chain reaction (PCR) in det ection 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 endophthalmi tis. 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 s amples (nine eyes) using restriction fragment length polymorphism (RFLP) an d DNA sequencing. RESULTS. A 100% concordance was obtained between PCR and culture-positive s amples. A PCR product was amplified from all 37 intraocular samples from ey es with suspected infection, whereas only 15 of 22 vitreous samples and 5 o f 15 aqueous samples were culture positive. Culture-negative PCR-positive s amples contained a preponderance of gram-negative bacterial sequences. Clon ing 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 a nd 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 clinic al signs of endophthalmitis. Gram-negative organisms seem to play a much mo re important role in the pathogenesis of this disease than previously thoug ht. PCR-based techniques have great value in the confirmation of the diagno sis of bacterial endophthalmitis especially in culture-negative eyes.