Ultrastructural demonstration of replicative herpes simplex virus type 1 transmission through corneal graft.

Abstract

Ultrastructural demonstration of replicative herpes simplex virus type 1 transmission through corneal graft Corneal graft is one of the most common transplant procedures in the world. Given the prevalence of herpetic keratitis in the population, the possibility of transmission of herpes simplex virus type 1 (HSV-1) remains a problem because it almost always results in rapid loss of graft transparency. Evidence of HSV-1 transmission via transplanted corneas has until now been indirect only, because the documented cases did not fully eliminate the hypothesis of reactivation of a virus in the recipient. Only Remeijer (1) demonstrated beyond doubt the genetic characterization of the same HSV-1 strain in both the donor eye and the recipient cornea in a case of graft failure. In Europe, corneas are typically stored in organ culture for 1 to 5 weeks at 31 to 37°C. Organ culture conditions promote viral replication in the cornea because they are similar to those of experimental herpetic co-cultures (2,3). The herpetic infections that go unnoticed during procurement can therefore be detected during storage by the appearance of massive necrosis of corneal endothelial cells caused by cytopathogenic effect (4). However, before our observation, there had never been a demonstration of active and asymptomatic replication during storage, especially one that resulted in transmission of a mature virus to the recipient. A 35-year-old woman with keratoconus underwent a transplantation with a cornea stored in organ culture for 9 days. Neither the 75-year-old donor nor the recipient had ever presented a clinical event of herpetic keratitis. The microscopic endothelial assessment performed at the end of organ culture showed a normal cell density and no cell mortality. Postoperative treatment consisted of 1% prednisolone eyedrops. Five days after grafting, an epithelial defect and an anterior uveitis developed and were consistent with herpetic infection. Despite acyclovir therapy, graft transparency was lost. Graft exchange was performed 8 months later with acyclovir. The second graft was uneventful. Polymerase chain reaction was positive for HSV-1 DNA in the failed graft and in the corresponding donor corneal specimen, which remained postoperatively (fixed for electron microscopy at the time of the first transplantation). Donor and recipient viral strain identity was confirmed using a polymerase chain reaction-based approach (5). Concomitantly, transmission electron microscopy of the donor specimen exhibited many HSV particles, located either within nuclei and cytoplasm of the keratocytes or as shedded enveloped particles, indicating mature and infectious viruses (Fig. 1). Figure 1: Sections viewed by transmission electron microscopy of the donor corneal specimen, resin-embedded and stored at the time of the first graft. Numerous icosahedral herpesviridae capsids were found in the keratocytes’ cytoplasm and nucleus (A and B). Some were empty (arrow), others full (arrowhead). Numerous mature, enveloped particles were found shedding from the cell, indicating replicative viruses (B, asterisk). Scale bar: 0.5 μm.Until now, HSV-1 transmission via transplanted cornea and its clinical manifestations prompted the assumption of reactivation of a latent virus borne by the graft, possibly triggered by surgical stress and amplified by postoperative steroids. Our observation provides another explanation. The transmission electron microscopy of the donor specimen indicates for the first time the presence of a mature and likely infectious virus within a cornea that showed no endothelial necrosis during the microscopic assessment performed before delivery by the eye bank. This emphasizes the possibility of a viral replication within the stored cornea during the organ culture period without any routinely detectable cellular changes in the graft. The mechanisms of such a smoldering viral infection have yet to be fully understood. Acknowledgments. The authors thank A. Blanc, I. Anselme, and J. Gouttesoulard of the Saint-Etienne electronic microscopy center for their technical assistance. Gilles Thuret Sophie Acquart Philippe Gain Gilles Thuret Jean-Marc Dumollard Chloé Manissolle Lydia Campos-Guyotat Philippe Gain Marc Labetoulle Thomas Bourlet