Ocular Fluid Work-up in Presumed Trematode Induced Granulomatous Uveitis

Patients with PTIGU of any age presenting to the Kasr Al-Aini Uveitis and Ocular Immunology Clinic who are scheduled to undergo AC wash or pars plana vitrectomy will be included in the study For all included patients, we will retrieve the age, gender, eye laterality, duration of disease, and the treatment received (topical, regional, and systemic). Careful review of systems and thorough investigations will be done as guided by clinical presentation (to exclude other causes of granulomatous uveitis). These investigations may be in the form of complete blood picture, stool and urine analysis, fluorescent treponemal antibody absorbent test, angiotensin-converting enzyme, tuberculin skin testing, toxocara antibodies, toxoplasma antibodies, and chest X-ray.

All patients will undergo thorough clinical examination including corrected distance visual acuity (CDVA), intraocular pressure (IOP) measurement, and ophthalmic findings with slit-lamp examination will be collected including the AC with associated granulomas, iris status regarding presence of synechiae, gonioscopic findings, lens status and vitreous inflammatory grades according to the Standardized Uveitis Nomenclature (SUN) classification. Dilated fundus examination with slit-lamp biomicroscopy and indirect ophthalmoscopy with scleral indentation will be explored to document the presence of retinal detachment, macula status, and grades of proliferative vitreoretinopathy (PVR). Also, B scan ultrasonography (US) will be done if media opacities precluded fundus examination. Ultrasonographic biomicroscopy (UBM) will be done to determine the presence and location of ciliary body granuloma.

All surgeries will be done under general anesthesia by the same experienced posterior segment surgeon. All eyes will receive topical antibiotics with topical prednisolone acetate 1% eye drops given every 2 hours for the first 48 hours followed by 5 times daily to be tapered according to AC reaction in addition to antibiotic combined with steroid ointment at bedtime.

Samples will be stored in sterile Epindorf tubes at -80°C. DNA will be extracted from the samples using DNeasy tissue kit (Qiagen, Hilden, Germany) according to manufacturer's.

A primer pair, CO1F (5-GATCGTA AATTTGGATACTGC-3) and CO1R (5-CCAACCATAAACATATGATG-3) targeting trematodal mitochondrial cytochrome c oxidase subunit 1 (CO1) gene will be used. PCR will be made in 25 µL reaction containing 12.5 µL MyTaq HS red Master Mix (Bioline, England), 0.5 µM of each primer and 0.5 µL DNA extract. PCR conditions will include initial denaturation at 95°C for 4 min followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 54°C for 15 s and extension at 72°C for 30 s. PCR products will then be examined on 1.5% agarose gel stained with ethidium bromide to detect the expected 250 bp amplified product. In samples undergoing sequencing, the amplified products will be loaded on the gel and purified using the DNA Purification Kit (Promega, Madison, WI, USA). The cyclic sequencing reaction will be performed. Samples will be denatured at 96°C for 2 min and then cycled 28 times at 96°C for 10 s, 52°C for 10 s, and 60°C for 4 min. Unincorporated nucleotides will be removed by using sodium carbonate, 125 mmol/L EDTA, and absolute alcohol. HI-Di Formamide (Applied Biosystems) will be used to stabilize the single-strand template before preforming sequencing. Sequences will be analyzed by using BLAST for species-level identification.