Pathogen identification and biomarkers characterization in aqueous and vitreous humor in infectious and non infectious uveitis

Abstract

Uveitis is a sight-threatening intraocular inflammatory disorder affecting mainly the uvea and that is estimated to be responsible for approximately 10 to 15% of the blindness in the United States. It is most common in working-age population. It may have an infectious or noninfectious etiology, associated or not with a systemic disease. In Brazil, infectious uveitis corresponds to approximately 43 to 63% of all uveitis cases in tertiary centers. The main infectious causes are toxoplasmosis, tuberculosis, syphilis, herpesviruses, human immunodeficiency virus, human T cell lymphotropic virus type I/II, among others. In noninfectious uveitis, there may be activation of innate immunity (autoinflammatory disease) or of adaptive immunity (autoimune disease), in response to an infectious or environment trigger, leading to tissue damage. Idiopathic cases, which correspond to 30 to 60% of uveitis cases, may remain without a specific etiology throughout the patient follow-up. Diagnosis is guided by the medical history of the patient and physical examination, further systemic and laboratory investigations are oriented by the anatomic characteristics of uveitis and by knowledge of varied disease patterns. A definite diagnosis is extremely rare since the changes mainly occur in loco and systemic aspects are usually nonspecific. Nevertheless, intraocular fluid analysis has had remarkable advancements in parallel to new molecular technologies. Thus, the present study aims to identify associated pathogens and to characterize the cytokine production profile in intraocular fluids of uveitis patients. More specifically, our goals are: a) apply efficient molecular methods in intraocular fluid to identify the causal agent of an infectious uveitis, using multiplex PCR, strip direct multiplex PCR and metagenomic next generation sequencing (mNGS); b) characterize an inflammatory and regulatory cytokine panel in aqueous and/or vitreous humor; c) correlate clinical outcomes with causal infectious agent and specific biomarkers. This prospective study will be conducted at a tertiary center and will include patients with active uveitis in a period of 6-12 months, previous to systemic treatment. Sample size will be 60 uveitis patients and 30 controls. Diagnosis work-up for uveitis etiology will be done as usual and included patients will be followed up for at least 12 months. Aqueous humor specimens (volume 100-200ul) sampled through anterior chamber paracentesis, and vitreous humor, through diagnostic (immunesuppression, malignancy, multiple infections, atypical evolution) or therapeutic vitrectomy (retinal detachment, vitreous hemorrhage), will be collected. Identification of uveitis-causing pathogens in intraocular samples will be carried out using solid phase direct multiplex strip PCR (for human herpes virus 1-6, toxoplasmosis, syphilis and HTLV-I/II) and real time PCR for toxoplasmosis and multiplex PCR (FTD Neuro 9® for herpesviruses); PCR negative samples collected from patients with a clinical diagnosis of infectious uveitis will be analysed by mNGS. Cytokine profile will be analysed using xmultiple analyte profile, Luminex® with a 41 cytokine/chemokine and a TGF-b plates. In patients with a definite diagnosis, a second aqueous humor sample will be collected for cytokine analysis. The main expected results are a) correlation between uveitis phenotype, causal agent and biomarkers in intraocular fluid, b) identification of potential novel causes of infectious uveitis through mNGS, and, c) establishment of solid base direct multiplex strip PCR for infectious uveitis diagnosis. (AU)