The Study Of Laboratory Diagnostic Methods For Intraocular Virus Uveitis In Immunocompetent Patients

ObjectiveFor immunocompetent patients,virus-associated intraocular inflammation could significantly affect visual effects and quality of life.The aims of this study were to describe the clinical characteristics in patients with viral anterior uveitis(VAU)and acute retinal necrosis syndrome(ARNS).We also examined aqueous humor(AH)proteome in VAU/ ARNS subjects and compared the proteome profile between VAU/ARNS and cataract patients in the hope of discovering a new biological diagnostic marker(s)with potential for new treatment strategy.Methods1、 Ninety-one AH samples were collected from 88 patients from November 2018 to November 2020 from the Uveitis & Ocular Immunology Service of Tianjin Medical University Eye Hospital(TMUEH):77 patients(79 eyes)with VAU,and11 patients(12 eyes)with ARNS.Patient demographic data such as age,sex,laterality,and systemic associations were documented.All study participants underwent a thorough ophthalmic examination,including evaluation of best-corrected visual acuity(BCVA),refractive error examination,slit-lamp biomicroscopy,intraocular pressure and ophthalmoscopy.AH samples were detected by q PCR to estimate the presence of herpes simplex virus(HSV),varicella-zoster virus(VZV),cytomegalovirus(CMV)and rubella virus(RV)DNA.Statistical analysis was performed on the obtained data.2、 From November 2018 to November 2020,twenty nine AH samples were collected from patients who had been diagnosed with VAU/ ARNS disease by q PCR,and sixteen cataract patients who underwent surgery were recruited as a control group.The protein of each sample was digested with trypsin and subsequently label-free quantitative proteomic analysis was performed to identify prognostic candidate proteins for protein-protein interaction networks and function enrichment analyses by Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways and gene ontology(GO).Results1、Among the VAU patients,twenty/79(25.3%)eyes underwent qPCR approaches for detection of virus DNA.The q PCR analysis showed positive results for HSV in 12/20(60%),for VZV in 3/20(15%),for CMV in 4/20(20%),and for RV in1/20(5%).VAU was diagnosed by PCR in AH,in 15 cases(75%),2、Of the VAU patients,depending on qPCR analysis were divided into two groups:positive result group comprised 20 eyes and control group,59 eyes with negative result.Sex and age distribution were similar between the two groups(p=0.855 and p=0.410,respectively).The positive result of q PCR was higher in patients with diease cause ≤3 months(P=0.029).Keratic precipitates(KP)(18/20,95% vs.39/59,90.6%;X2 test P=0.039),iris hypopigment(8/20,40% vs.8/59,13.6%;X2test P=0.026),iris atrophy(6/20,30% vs.2/59,3.4%;X2 test P=0.003),iris synechia(9/20,45% vs.4/59,6.8%;X2 test P<0.001),and elevated intraocular pressure(IOP)(15/20,75% vs.27/59,45.8%;X2 test P=0.024)were significantly more common in eyes with positive result of q PCR compared to q PCR-negative eyes.Iris synechia and diease cause ≤3 months were the most significant predictors of q PCR-proven infections.3、No statistically significant differences were found in clinical characteristics between the patients with and without recurrence.When we compared different pathogens,VZV had the highest IOP(41mm Hg,range 31-46 mm Hg)in q PCRpositive infections.High IOP was found in 53.2%(42/79)of the eyes and the mean IOP was 32.54±6.82 mm Hg.Mutton-fat KP and pigmented KP were significantly more frequently observed in eyes with high IOP(P=0.005,0.050),while macular edema and medium-sized KP were more common in normal IOP group(P=0.042,0.001).4、All patients with suspected ARNS underwent qPCR in AH.Result for viral DNA from HSV was positive in one patient,and VZV was positive in 11 patients.At the initial visit,the mean log MAR best-corrected visual acuity(BCVA)was 0.86±0.96,and the mean IOP was 24.21±11.03 mm Hg.In the early stage,anterior chamber cells and inflammatory vitreous opacity are common ocular findings mainly occurring in all patients.Other ocular clinical manifestations showing were yellow-white lesions in the peripheral retina(91.7%),retinal arteritis(66.7%),optic disc edema(58.3%),elevated IOP(58.3%),retinal phlebitis(50%),KP(50%),epiretinal membrane(50%),ciliary hyperemia(41.7%),and retinal hemorrhage(41.7%).5、A total of 359 proteins were noted and 186 differentially expressed proteins(DEPs)were identified in AH from the 45 patients.6、There were 85 DEPs between VAU group and cataract group.Of these proteins,41 differentially expressed proteins were detected to be down-regulated and 44 proteins were up-regulated in the AH from VAU patients compared to controls.The significantly up-regulated proteins were IGHG4,IGHM,P0DOX7,C1 QC,PGLYRP2,ACTB,IGHV1-2,IGLC2,CTSB and IGHA1,whereas DEFA1,LTBP2,WIF1,RBP3,PTGDS,CRYGS,CLSTN1,SPON1,SPOCK1 and SERPINI1 reduced most distinctly.7、There were 93 DEPs between ARN group and cataract group.Of these proteins,50 differentially expressed proteins were detected to be down-regulated and 43 proteins were up-regulated in the AH from ARN patients compared to controls.The significantly up-regulated proteins were IGHG4,IGLC2,IGHV3-49,IGKV1D-33,IGHV1-2,IGHM,P0DOX7,PLTP,ACTB and IGHV5-51,whereas CRYGS,PTGDS,WIF1,SOD3,SERPINI1,CLSTN1,CPAMD8,DEFA1,NBL1 and CUTA reduced most distinctly.8、There were 41 DEPs between ARN group and VAU group.Of these proteins,33 differentially expressed proteins were detected to be down-regulated and 8proteins were up-regulated in the AH from ARN patients compared to controls.The significantly up-regulated proteins were PLTP,IGKV1D-33,IGHV3-49,IGLC2,GAPDH,YWHAZ,IGHV1-69 and IGHV3-72,whereas CRYGS,SOD3,PTGDS,CPAMD8,SERPINI1,NPC2,WIF1,CUTA,NBL1 and CPE reduced most distinctly.Conclusion1、There are several similar clinical characteristics which makes a diagnosis of the correct virus infection uncertain.Therefore,it is useful to analyze viral DNA in AH to determine the appropriate treatment in the early stage of disease.2、The DEPs related to VAU/ARNS are mainly involved in complement and coagulation cascades,and cholesterol metabolism.The expression of Apolipoprotein A-I(APOA1)was significantly up-regulated in AH in VAU/ARN,while some other proteins such as Apolipoprotein E(APOE),Amyloid-like protein 2(APLP2),and Cystatin C(CST3)were down-regulated.The differentially expressed proteins in AH were related with the pathogenesis of VAU/ARNS.