The PCR assay was validated for vitreous fluid. Vitreous fluid from four patients was spiked with an internal plasmid control to evaluate the efficiency of DNA recovery and assess for the presence of PCR inhibition. Each vitreous sample tested positive for the plasmid DNA with no evidence of inhibition and negative for HHV-6 and HHV-7 DNA. Each sample was also tested for a cellular gene, beta-globin, and each was positive with values ranging from 10 cellular equivalents/ml of vitreous fluid to 1,754 cellular equivalents/ml of fluid. Another aliquot of each vitreous sample was spiked with HHV-6 and HHV-7 infected cell lysate, and PCR of the vitreous sample was positive for both viral DNAs. Thus, these results indicated that DNA could be extracted successfully from vitreous fluid, the fluid did not inhibit the PCR assay, and that HHV-6 and HHV-7 DNA could be detected by PCR in vitreous fluid.
PCR was performed for CMV DNA using coded vitreous samples from 33 patients that had been stored for several years at −80°C, including 11 samples that had previously tested positive for CMV DNA at the time they had been obtained, to determine if herpesvirus DNA was stable after prolonged storage. The other 22 samples were eyes of patients without inflammatory ocular disease (e.g vitrectomy samples from diabetics, vitreous fluid from retinal detachment repairs, intraocular melanoma). One of these 22 samples which had initially tested negative for CMV DNA was CMV DNA positive in the assay. It is unclear if the positive sample had been incorrectly assayed as negative when tested originally or if the extraction and PCR procedures had improved in the 15 years since the sample was retested. Three samples which had initially tested negative gave faint signals which were below the level of the standard curve (<250 copies of CMV/ml). Ten of the 11 known CMV DNA PCR positive samples were again positive for CMV DNA PCR with 933 to >250,000 CMV DNA copies/ml (median 52,000 CMV DNA copies/ml); the one negative sample had been positive using one set of primers to the CMV major IE gene, but negative using another set of primers to the sample CMV gene when originally tested. These results indicated that herpesvirus DNA could still be detected by PCR in the stored samples.
An additional 68 vitreous samples were obtained from patients with non-inflammatory ocular disease (9 patients) ocular inflammation including patients with retinitis, vitritis, iritis (58 patients), and lymphoma (1 patient). Of these 68 samples, 6 were known to be CMV DNA positive, 1 was known to be HSV DNA positive, and 10 were from patients with HIV.
Of the 101 total vitreous samples, one was positive for HHV-6A DNA, 1 for HHV-6B DNA, and none for HHV-7 DNA (). The HHV-6A DNA positive sample was from a patient with a detached retina and CMV retinitis. The sample contained 4,950 HHV-6A DNA copies per ml, was positive for CMV DNA (32,000 copies per ml), and negative by for HHV6-B, HHV-7, HSV, and VZV DNA by PCR. Based on detecting 33,000 human β-globin DNA copies per ml in the vitreous sample, there were approximately 0.30 copies of HHV-6A per cellular genome. The HHV-6B DNA positive sample was from a patient with idiopathic ocular inflammation and contained 10,140 HHV-6B DNA copies per ml and was negative for HHV-6A, HHV-7, HSV, CMV, VZV, and toxoplasmosis DNA by PCR. Based on the 22,000 human β-globin DNA copies per ml in the vitreous fluid, there were an estimated 0.92 copies of HHV-6B per cellular genome.
HHV-6A and HHV-6B DNA positive vitreous fluid samples from 101 total samples