The incidence and influence of the donor corneas positive for herpesviridae DNA in keratoplasty

The information of 569 donors was registered on the unified organ donation website from April 2017 to July 2019, and informed consent was obtained from all participants and/or their legal guardians. Tests for HBV, HCV, syphilis, and HIV were all negative before organ donation. Under sterile conditions, a suitable trephine was used to strip a 3- to 4-mm section of the sclera and posterior cornea within 12 h after donor death. Then, corneoscleral buttons were obtained and stored in Optisol-GS (Bausch & Lomb, Irvine, CA, USA) at 4 °C. The endothelial cell density (ECD) of all donor corneas was quantified by a certified technician at our eye bank using an EB-3000 XYZ eye bank specular microscope (HAI Laboratories Inc., Lexington, MA, USA). The study was performed according to the tenets of the Declaration of Helsinki and was approved by the local ethics committee.

In this study, all procedures were performed by an experienced surgeon (Jing Hong) at Peking University Third Hospital. The donor circular limbal corneas comprising the corneal endothelium were obtained during surgery with aseptic precautions and immediately delivered to the laboratory for virological analyses. The samples were obtained during consecutive cornea transplant procedures. All samples were tested for HSV-1, HSV-2, VZV, CMV, and EBV DNA.

We extracted DNA from the corneal tissues using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. In brief, the corneal rims were cut into small pieces, placed in a 1.5-mL microcentrifuge tube, and digested with ATL buffer and proteinase K. The extracted DNA was diluted in water; a total of 50 ng was subjected to PCR.

HSV-1, HSV-2, VZV, CMV, and EBV were detected using qualitative commercial, TaqMan-based methods (HSV-1/HSV-2 Typing Real-Time PCR Kit Z-SD-0136-02, VZV Real-Time PCR Kit OD-0024-02, CMV Real-Time PCR Kit Z-OD-002-02, and EBV Real-Time PCR Kit Z-OD-0023-02, Liferiver Bio–Tech Corp., China), in accordance with the manufacturer’s instructions. Real-time PCR assays were performed using reagents from PE Biosystems (PE Applied Biosystems, Foster City, CA). The limit of detection for all herpesviridae DNA was 10 copies/μg. Each sample was processed with the addition of an internal control to assess isolation and amplification efficacy. Positive and negative controls, as well as internal controls, were provided by the kit manufacturers.

The clinical outcome of transplantation was assessed by best-corrected visual acuity (BCVA), intraocular pressure (IOP), ECD, and complications at 1 and 3 months postoperatively. The endothelial cell (EC) loss rate was calculated according to the ECD. The ECD of the central area was measured by in vivo confocal microscopy (IVCM; HRT III: Heidelberg Engineering, Heidelberg, Germany). Graft attachment was assessed with anterior segment optical coherence tomography (AS-OCT; Carl Zeiss Meditec, Dublin, CA, USA). The same certified ophthalmic technician performed all postoperative tests in patients using the same microscope.

In total, 942 donor cornea buttons were included in this study (Table 1). A total of 569 donors (286 males and 283 females), ranged from 0.17 to 88 years old (mean age 46.8 ± 17.7 years). A total of 373 donated both corneas, and 196 donors donated a single cornea. The most common causes of death in donors were trauma or accident in 48.2% (n = 274), cancer in 29.2% (n = 166), cardiovascular disease in 15.6% (n = 89), respiratory failure in 5.1% (n = 29), and other in 1.9% (n = 11). The mean death to preservation duration was 6.7 ± 3.2 h (range, 0–12 h), and the mean death to surgery duration was 5.3 ± 2.6 days (range, 1–11 days). A total of 403 donors were in-hospital patients (403/569 = 70.8%). The mean ECD was 3239 ± 440 cells/mm² (range, 2016–6201 cells/mm²).

Twenty-three donor cornea buttons were virus-positive according to RT-PCR (Table 1). Of the 22 cornea donors (10 males and 12 females), ranging from 0.47 to 70 years old (mean age 44.2 ± 17.1 years), the most common donor cause of death was cancer in 54.5% (n = 12), trauma or accident in 18.2% (n = 4), cardiovascular disease in 18.2% (n = 4), respiratory failure in 4.5% (n = 1), and other in 4.5% (n = 1). The mean death to preservation duration was 6.9 ± 2.5 h (range, 2–11 h), and the mean death to surgery duration was 4.5 ± 2.3 days (range, 1–9 days). Twenty donors were in-hospital patients (2/22 = 90.9%). The mean ECD was 3381 ± 463 cells/mm² (range, 2722–4735 cells/mm²).

Donor age, sex, ECD, time from death to the preservation, and time of death to surgery were not significantly different between the groups (t = 0.76, P₁ = 0.448; χ² = 0.212, P₂ = 0.645; t = −1.562, P₃ = 0.119; t = −0.227, P₄ = 0.782; t = 1.541, P₅ = 0.124, respectively). The cause of donor death and inpatient status was significantly different between the groups (χ² = 10.235, P₆ = 0.037 < 0.05; χ² = 4.467, P₇ = 0.035 < 0.05, respectively) (Table 1).

The total donor cornea herpesviridae DNA positivity rate was 2.44% (23/942). The positivity rate of CMV, HSV-1, VZV, and EBV DNA were 34.78% (8/23), 30.43% (7/23), 26.09% (6/23), and 8.7% (2/23), respectively. HSV-2 DNA was not detected in this study.

Twenty-seven recipients (4 herpesviridae-positive grafts were used in DALK and DSAEK at the same time) who received the virus-positive donor corneas were included in this study (Table 2). The ages of the 27 recipients (19 males and 8 females) ranged from 0.33 to 86 years (mean age 38.1 ± 26.0 years). The diagnoses of the recipients are listed in Table 2. Nine recipients (9/27 = 33.3%; HSV-1/CMV/VZV/EBV = 1/5/3/0) underwent penetrating keratoplasty (PK), 11 recipients (11/27 = 40.7%; HSV-1/CMV/VZV/EBV = 5/2/2/2) received corneal endothelium transplantation, and 7 recipients (7/27 = 25.9%; HSV-1/CMV/VZV/EBV = 4/2/1/0) underwent deep anterior lamellar keratoplasty (DALK).

The follow-up time was 3 months. AS-OCT revealed that the attachments of corneal endothelial grafts were good. Recipients Nos. 22, 23, 24, and 25 received a pair of HSV-1-positive corneas from a 50-year-old male who died in a traffic accident. Analysis of this donor’s records did not reveal any infectious diseases, and he was admitted to the intensive care unit (ICU) for 1 week before donation. Recipients Nos. 23 and 25 were both young males with keratoconus, and they underwent DALK on the same day. The transparent corneas remained completely transparent throughout the 3-month follow-up period. Recipients Nos. 22 and 24 were 81-year-old female and 54-year-old male, respectively, and both suffered from bullous keratopathy due to phacoemulsification. They underwent corneal endothelium transplantation on the same day. Recipient No. 22 showed signs of herpesviridae infection 3 days after surgery and received intraocular injections with ganciclovir twice within 4 days (aqueous humor tested HSV-1-positive) (Fig. 1b, c). Unfortunately, recipient no. 24 also showed signs of virus infection 6 days after surgery and received intraocular injection with ganciclovir twice within 14 days (aqueous humor-tested HSV-1-positive) (Fig. 2b, c). Despite an appropriate and intensive treatment course of parenteral and local therapies, the transplanted tissue did not maintain good function (Figs. 1d and 2d). It was necessary to perform Descemet’s stripping automated endothelial keratoplasty (DSAEK) half a month later (Figs. 1e and 2e). During the second surgery, the aqueous part of the removed graft and the donor corneoscleral tissue were obtained for PCR analysis; the other part of the graft was examined with transmitted electron microscopy (TEM). The aqueous and removed grafts were all positive for HSV-1 DNA, and the new donor was negative for herpesviridae DNA. TEM findings showed only denuded Descemet’s membrane (DM) without any endothelial cells left on the graft of No. 22; viral particles were spotted within the posterior stroma of the DSAEK graft of No. 24 by TEM. The grafts of recipients 22 and 24 remained transparent after retransplantation at the 6-month follow-up (Figs. 1f and 2f). This study had already been published [11].

The mean ECD of the grafts (except recipients who received DALK and recipients Nos. 22 and 24) was 2543 ± 561 cells/mm² (n = 18; range, 1387–3452 cells/mm²) at 3 months postoperatively, representing a mean postoperative EC loss of 25.19 ± 16.25% (range, 2.39–60.71%). The EC loss in HSV-1-, CMV-, VZV-, and EBV-positive recipients was 24.40%, 32.86%, 15.57%, and 24.21%, respectively, and there was no significant difference between recipients with those viruses (F = 1.073, P = 0.392). The EC losses in those who underwent PK and DSAEK were 23.80% and 26.58%, respectively, and there were no significant differences between the two transplantation procedures (t = −0.344, P = 0.736). There was no correlation between IOP and EC loss (P = 0.934).