Title : Low-volume direct multiplex PCR for etiological diagnosis of infectious uveitis
Abstract:
Purpose: To describe the pathogens detected in intraocular fluid of individuals with active uveitis using a low-volume direct multiplex PCR.
Methods: Eighty-six individuals with active uveitis were included in this study between July, 21 and November, 23. All participants had at least (2+) cells in anterior chamber (AC) or vitreous haze. Samples were obtained by AC paracentesis (81 samples) or pars plana vitrectomy (5 samples). We also included 23 control samples, being aqueous humor from cataract surgery (21 samples) and vitreous humor from epiretinal membrane surgery (2 samples). Twenty µl of the sample was analysed through a qualitative direct multiplex real-time polymerase chain reaction (PCR) assay, developed by Japanese researchers for uveitis diagnosis purpose. It detects herpes simplex virus (HSV) 1 and 2; varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV6), human T-lymphotropic virus (HTLV), Treponema pallidum and Toxoplasma gondii. This multiplex PCR was validated mainly in Japan. This study was approved by Institution Ethics Committee (CAAE 91413918.5.0000.0068) and all participants signed the informed consent form.
Results: The clinical characteristics of the uveitis were as follows. Anatomic classification were mainly anterior (39 patients; 45.3%) and posterior (37 patients; 43%). The onset/course of the uveitis: first acute episode in 32 patients (37.2%), recurrent in 28 patients (32.5%) or chronic in 29 patients (33.7%). Overall positivity was 23.5% (20/86 samples); among acute uveitis, it was 28.8% (17/59 samples) and, among infectious uveitis (diagnosis before intraocular fluid analysis), it was 35.1% (20/57 samples). Herpes virus was detected in 10 samples (50%). The agent detected and final diagnosis based on clinical and PCR results were: EBV in three samples (VZV PCR+ acute retinal necrosis and coinfection with EBV; PCR- toxoplasmosis but EBV+ in two cases); CMV (CMV associated anterior uveitis in two cases) and HHV6 (HHV6 associated anterior uveitis in two cases) in two samples, each; VZV (VZV associated acute retinal necrosis), HSV2 (HSV2 associated acute retinal necrosis) and HSV1 (HSV1 associated acute retinal necrosis) in one sample each. Toxoplasma gondii was detected in 9 samples (45%) and all cases had retinochoroiditis. HTLV and T. pallidum were detected in one sample each and both cases had diffuse uveitis. The strip PCR changed the etiological diagnosis in five cases (5.8%; 3 cases of herpetic uveitis to toxoplasmosis, one case of syphilis to toxoplasmosis and one case of toxoplasmosis to herpetic uveitis).
Conclusion: For uveitis etiological diagnosis, direct strip PCR was able to demonstrate the infectious agent in almost one fourth of our sample, using just a small sample volume, remaining material for further analysis of this valuable low-volume sample. The multiple pathogens selected encompass the most important infectious uveitis etiologies. Further, even though most patients were under use of topical and/or systemic therapy, we could not observe interference of treatment in PCR positivity.
