Objective:To investigate the virological testing results of patients with corneal graft at the time of repeat keratoplasty and the diagnostic efficacy of multiple viral examinations.Methods:A case-control study was conducted.A total of 14 consecutive patients diagnosed with corneal graft failure were enrolled as graft failure group from March 2018 to December 2018 in Peking University Third Hospital, and 15 consecutive patients diagnosed with bullous keratopathy (BK) were enrolled as BK group in the meantime.All patients had unilateral involvement and indications for keratoplasty.Serum samples were collected from venous blood on the day of surgery, and specimens of aqueous humor and corneal tissue were obtained during corneal transplantation.Viral DNA in aqueous humor and corneal specimens was detected by real-time polymerase chain reaction (PCR).The level of viral antibodies in serum and aqueous humor was determined by enzyme-linked immunosorbent assay and the Goldmann-Witmer coefficient (GWC) was calculated.The tested viral species included herpes simplex virus (HSV), herpes zoster virus (VZV), and cytomegalovirus (CMV).For graft failure group, the relevance between elevated intraocular pressure, multiple previous keratoplasty surgeries, histories of viral keratitis and any positive result of viral analyses in this study were measured by the kappa consistency test.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Peking University Third Hospital (No.2017299-02).Written informed consent was obtained from each subject.Results:At the time of keratoplasty, 9 out of 14 eyes in the graft failure group tested positive for at least one type of virus, with 6 eyes positive for CMV and 3 eyes positive for VZV.Positive aqueous humor PCR analysis detected VZV in 5 out of 14 eyes.Corneal tissue PCR analysis detected CMV in 5 out of 14 eyes.Positive GWC calculations detected CMV in 3 out of 10 eyes.The concordance between viral DNA and antibody detection was poor.All eyes in BK group were negative for the virological test, except for 2 eyes (2/15) with elevated aqueous humor GWC for CMV.The prevalence of viral infection was 64.2%(9/14) in graft failure group, which was significantly higher than 13.3%(2/15) in BK group ( P=0.014).In graft failure group, 7 eyes (7/14) had elevated intraocular pressure, 3 eyes (3/14) had multiple keratoplasty surgeries, and 6 eyes (6/14) had viral keratitis before this keratoplasty.However, none of these factors showed significant relevance with positive virological results (kappa=0.143, -0.155, -0.286). Conclusions:Viral infection has become a major cause of corneal graft failure.A combination of various virological analyses during keratoplasty can help to clarify the etiology and the viral infection status, and ultimately guide subsequent treatment.