Validation of a diagnostic multiplex polymerase chain reaction assay for infectious posterior uveitis

Objective: To valide a multiplex polymerase chain reaction (PCR) assay capa ble of simultaneously screening vitreous biopsy specimens for a panel of co mmon pathogens in posterior uveitis. Methods: A multiplex PCR assay using novel primer sets for cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Toxopl asma gondii was developed. The sensitivity of the assay was determined for purified pathogen DNA. Twenty-one vitreous specimens from patients with pos terior uveitis were tested by both multiplex and monoplex PCR. Results: Fewer than 10 genomes of VZV and fewer than 100 genomes of HSV, CM V, and T gondii could be detected using the new primer sets. When used in m ultiplex, the assay lost less than 1 log of sensitivity. Monoplex PCR detec ted pathogen DNA in 18 of 21 patient samples, multiplex PCR detected pathog en DNA in 15 of the 18 samples positive by monoplex PCR. None of 10 negativ e control samples were positive for pathogen DNA. Conclusions: Multiplex PCR has adequate sensitivity to simultaneously scree n a substantial differential diagnosis for posterior uveitis in a single re action, without loss of specificity. This assay may reduce the time and cos t involved in PCR-based molecular diagnostics of infectious pathogens. Clinical Relevance: Mutiplex PCR may allow rapid diagnosis of infectious po sterior uveitis.