Purchase this article with an account.
T-K Chan, X Sun, S-P Chee, C Chee, K-P Song; Nested PCR and Real-time PCR Applications in Rapid Diagnosis of Infectious Endophthalmitis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4432.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate the use of nested polymerase chain reaction (nested-PCR) and real-time PCR in the diagnosis of infectious endophthalmitis. Method: A nested PCR protocol, which allowed rapid detection of bacteria or fungi implicated in infectious endophthalmitis, was developed. This enabled identification of the pathogens at three different levels, namely Gram type level (Gram positive or Gram negative), genus level (Staphylococci or streptococci) and species level (Streptococcus pneumoniae, Group B Streptococci, Propionibacterium acnes, Pseudomonas aeruginosa, Mycobacterium tuberculosis). This protocol was first tested on 33 bacterial or fungal species (7 ATCC strains and 43 ocular isolates) covering a range of common ocular pathogens and they served as positive controls. The optimized protocol was then applied to 26 ocular samples (vitreous-23, aqueous-3) obtained from patients with clinically suspected endophthalmitis. Thirty vitreous samples from non-infected eyes served as negative controls. Real-time PCR was also performed on 12 of the 26 clinical vitreous samples. Results: All ocular isolates and standard strains could be correctly identified at the Gram type, genus, or species level and as few as one single organism could be detected by nested PCR. All the 30 negative controls from non-infected eyes showed the absence of any microorganism. Among the 26 vitreous and aqueous samples, 5 were positive by both culture and nested PCR and two PCR-negative samples were also proven negative by culture. Nineteen clinical samples were positive only by PCR. Therefore, PCR detected potential pathogens in 92% (n=24) of the infectious samples as compared to 19% (n=5) by culture. This difference was statistically significant (McNemar's test: P<0.001). Real-time PCR showed high concordance (94%) with normal nested PCR. The duration taken for nested PCR and real-time PCR were 6 and 5 hours respectively. Conclusion: In this study, nested PCR was more sensitive and rapid for the detection and identification of pathogens in patients with suspected endophthalmitis, as compared to the conventional culture technique. Real-time PCR is a potentially useful diagnostic technique in view of its quantitative nature and rapid capability.
This PDF is available to Subscribers Only