Validation of a Diagnostic Multiplex Polymerase Chain Reaction Assay for Infectious Posterior Uveitis

Abstract

To valide a multiplex polymerase chain reaction (PCR) assay capable of simultaneously screening vitreous biopsy specimens for a panel of common 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 Toxoplasma gondii was developed. The sensitivity of the assay was determined for purified pathogen DNA. Twenty-one vitreous specimens from patients with posterior uveitis were tested by both multiplex and monoplex PCR. Results: Fewer than 10 genomes of VZV and fewer than 100 genomes of HSV, CMV, and T gondii could be detected using the new primer sets. When used in multiplex, the assay lost less than 1 log of sensitivity. Mono-plex PCR detected pathogen DNA in 18 of 21 patient samples; multiplex PCR detected pathogen DNA in 15 of the 18 samples positive by monoplex PCR. None of 10 negative control samples were positive for pathogen DNA. Conclusions: Multiplex PCR has adequate sensitivity to simultaneously screen a substantial differential diagnosis for posterior uveitis in a single reaction, without loss of specificity. This assay may reduce the time and cost involved in PCR-based molecular diagnostics of infectious pathogens. Clinical Relevance: Mutiplex PCR may allow rapid diagnosis of infectious posterior uveitis.

Multiplex polymerase chain reaction for the detection of herpes simplex virus, varicella-zoster virus and cytomegalovirus in ocular specimens

“…A number of studies have reported utility of multiplex PCR in diagnosis of intraocular inflammation. 7,13 In one study, multiplex PCR for six herpesviruses has been elegantly used with "Stair primers" on tear samples from patients with keratitis, conjunctivitis and chronic dry eye. 14 To the best of our knowledge, these viruses have not been tested for, in multiplex PCR, on corneal scrapings.…”

“…Data presented here highlights the application of this versatile technique on diverse type of ocular samples, most notably corneal scrapings. Although a number of studies have reported utility of multiplex PCR in diagnosis of intraocular inflammation, 7,13 to the best of our knowledge, multiplex PCR developed for this group of viruses has not been tested and reported earlier for corneal scrapings and either fixed or fresh ocular tissues.…”

Treatment outcome and risk factors for visual loss in Cytomegalovirus endotheliitis

“…Real-time polymerase chain reaction was also performed in the later cases [15]. The in-house nested multiplex PCR test was developed based on the method described by Dabil [16], which simultaneously detects Herpes simplex virus (HSV), Varicella zoster virus (VZV), CMV, and Toxoplasma gondii (T. gondii) DNA. The lower limits of detection for HSV1, HSV2, CMV, VZV, and T. gondii are 360, 120, 50, 40, and three copies per PCR reaction respectively, with a sensitivity and specificity of >95%.…”

Prospective comparison between conventional microbial work-up vs PCR in the diagnosis of fungal keratitis

“…In ophthalmology, the use of PCR in the diagnosis of causative pathogens in uveitis, [8][9][10][11] and viral keratitis, has increased in the past few years. 12 However, its application in microbial keratitis has been limited.…”