Low-volume direct strip multiplex PCR of intraocular fluid in uveitis

Abstract

Purpose : To analyse the usefulness of low-volume direct multiplex PCR of intraocular fluid for the etiological diagnosis of uveitis.

Methods : This prospective study is part of a project to identify the pathogen in ocular fluid in uveitis which included direct strip multiplex PCR (part 1), metagenomic (part 2) and cytokine analysis (part 3). Part 1 of the study is described here. Individuals, all with active uveitis (cells in anterior chamber ≥ 2+, SUN 2005), were included from July 21 to Nov 22, after obtaining informed consent. Samples were obtained by AC paracentesis (38 samples) or pars plana vitrectomy (2 samples). Twenty µl of sample was analyzed using a direct multiplex qualitative polymerase chain reaction (PCR) assay (Applied Biosystems 7500 Fast Real-Time PCR systems), developed by Japanese researchers for uveitis diagnosis purpose. It detects herpes simplex virus 1 and 2; varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human herpes virus 6, human T-lymphotropic virus, Treponema pallidum and Toxoplasma gondii. The mean assay time was 58 minutes and it detects 25 copies/well. This multiplex PCR was validated mainly in Japan (Nakano 2021), .Aqueous humor samples of two individuals with cataract were included.

Results : A total of 40 samples were collected from 40 individuals with active uveitis (Table 1). Uveitis were anterior or posterior in 18 (45%) and 17 (42.5%) patients, respectively. It was the first acute episode, a recurrence or chronic in 16, 9 and 15 individuals, respectively. Overall positivity was 22.5% (9/40), 32% (8/25) for acute/recurrent uveitis vs 6.7%(1/15) for chronic uveitis. Among suspected infectious uveitis the positivity was 30% (9/30). Herpes virus was detected in 4 samples: 2 for HHV6 (anterior uveitis), 1 for HSV2 (acute retinal necrosis) and 1 for EBV (neuroretinitis); T. gondii was detected in 4 samples (retinochoroiditis) and T. pallidum in another sample (panuveitis) (Table 2). The strip PCR changed the etiological diagnosis in two cases (5%; herpetic uveitis or syphilis to toxoplasmosis) and showed an unexpected herpesvirus as causative agent in another two cases.The two controls were PCR negative.

Conclusions : For uveitis etiological diagnosis, direct strip PCR was able to demonstrate the infectious agent in one third of our sample with the unique characteristics of using very small sample volume, of testing for multiple pathogens all together and for a rapid results.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.