Detection and quantification of pathogenic bacteria and fungi using real-time polymerase chain reaction by cycling probe in patients with corneal ulcer.
OBJECTIVE
To detect and quantitate the causative pathogens in patients with corneal ulcer using real-time polymerase chain reaction (PCR) by cycling probe.


METHODS
Clinical and laboratory study of 40 eyes of 40 patients diagnosed with corneal ulcer. Two methods were used for pathogen detection: bacterial culture and real-time PCR with the patient's corneal scrapings. Probes and primers of real-time PCR were designed to be pathogen specific for simultaneous detection of Staphylococcus aureus, Staphylococcus pneumoniae, Pseudomonas aeruginosa, methicillin-resistant S aureus, Candida species, and Fusarium species. Results by both methods were evaluated and compared.


RESULTS
Of 40 eyes, 20 eyes had the same pathogens detected by both methods and those were S aureus (3 eyes; mean [SE], 3.8 [1.3] x 10(1) copies/sample), S pneumoniae (5 eyes; mean [SE], 5.6 [5.1] x 10(3) copies/sample), P aeruginosa (8 eyes; 5.1 [4.0] x 10(3) copies/sample), methicillin-resistant S aureus (1 eye; 1.0 x 10(2) copies/sample), and Candida species (3 eyes; mean [SE], 8.8 [4.9] x 10(3) copies/sample). Six eyes showed negative results by both methods. Results of both methods disagreed in 14 eyes; specifically, 11 had positive PCR results only, 2 had positive culture results only, and 1 eye had positive results for different pathogens.


CONCLUSIONS
The real-time PCR assay can simultaneously detect and quantitate bacterial and fungal pathogens in patients with corneal ulcer. Real-time PCR can be a fast diagnostic tool and may be useful as an adjunct to identify potential pathogens.
Itahashi
Motoki
M
Department of Ophthalmology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Japan.
Higaki
Shiro
S
Fukuda
Masahiko
M
Shimomura
Yoshikazu
Y
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D015373:Bacterial Typing Techniques / Q000379:methods; D003320:Corneal Ulcer / Q000175:diagnosis* / Q000382:microbiology; D017931:DNA Primers; D015342:DNA Probes; D015818:Eye Infections, Bacterial / Q000175:diagnosis* / Q000382:microbiology; D015821:Eye Infections, Fungal / Q000175:diagnosis* / Q000382:microbiology; D005260:Female; D005658:Fungi / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008297:Male; D008875:Middle Aged; D016533:Mycological Typing Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D055815:Young Adult
D017931:DNA Primers; D015342:DNA Probes