Objective: To validate a real-time polymerase chain reaction (PCR) assay allowing rapid and sensitive detection and quantitation of 4 common infectious posterior uveitis pathogens.
Methods: A real-time PCR assay using previously validated primer sets for cytomegalovirus, herpes simplex virus, varicella-zoster virus, and Toxoplasma gondii was developed. A standard curve for quantitation of pathogen load was generated for each pathogen using SYBR Green I fluorescence detection. Ocular samples from patients with posterior uveitis and from negative control samples were assayed and compared with standards to identify pathogens and quantify infectious load.
Results: Sensitivity for detection of purified pathogen DNA by PCR was not reduced by application of the real-time method. Standard curves for the quantitation of pathogen loads showed sensitivity to fewer than 10 organisms for all pathogens. The technique was applied to 2 clinical problems. First, sensitivities of existing monoplex and multiplex PCR were compared by real-time PCR. No significant difference in sensitivity was observed between multiplex and monoplex techniques. Second, pathogen loads of vitreous specimens from patients previously diagnosed as having infectious posterior uveitis were calculated. Pathogen loads were found to be generally higher for patients with disease caused by varicella-zoster virus than those caused by cytomegalovirus or herpes simplex virus.
Conclusions: Real-time PCR may be applied to infectious agents responsible for posterior uveitis. This technique will likely prove useful for the diagnosis of posterior uveitis as well as the linkage of pathogen to disease.
Clinical relevance: Real-time PCR provides a rapid technique for quantitatively evaluating ocular samples for the presence of infectious pathogens.