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Clinical Sciences

Detection of Hepatitis C Virus in the Corneas of Seropositive Donors

Lee, Hung Ming M.B., B.S., M.Med.(Ophth.), F.R.C.S.(Ed.), F.A.M.S.(Ophth.); Naor, Joel M.D.; Alhindi, Ramiz M.D., F.R.C.S.C.; Chinfook, Trevor M.D., F.R.C.S.C.; Krajden, Mel M.D., F.R.C.P.C.; Mazzulli, Tony M.D., F.R.C.P.C.; Rootman, David S. M.D., F.R.C.P.C.

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Transmission of diseases from donor tissues to recipients is one of the potential hazards associated with all types of organ and tissue transplantation. Medical standards for selection of donor corneal tissues have been developed by the Eye Bank Association of America (EBAA) and the United States Food and Drug Administration to ensure that donor corneas are of the highest quality and that the donor is free of transmissible diseases. 1,2 The EBAA presently requires that all potential donors be serologically tested for human immunodeficiency virus types 1 and 2, hepatitis B surface antigen, and hepatitis C virus (HCV). The only systemic infectious diseases documented to be transmitted by corneal transplantation so far include hepatitis B virus, Creutzfeldt-Jakob disease, and rabies. 3–7

Although there have been no reported cases of HCV transmission through corneal transplantation, the EBAA adopted HCV antibody testing in 1991 and all potential donors must have a negative screening or confirmatory test for HCV. The current standard screening technique for HCV is the second-generation enzyme immunoassay (EIA), versions of which are produced by Abbott Diagnostic (Abbott Laboratories, Abbott Park, IL, U.S.A.) and Ortho Diagnostic (Ortho Diagnostics, Raritan, NJ, U.S.A.) under the designation HCV EIA 2.0. These assays test for serum antibodies to multiple HCV antigens. Positive results may be confirmed by commercially available immunoblot systems, such as second-generation radio-immunoblot assay (RIBA-II) or Matrix-HCV, which are considered to be more sensitive to these antigens. Using immunoblot as a reference, Laycock et al. 8 demonstrated that the second-generation EIA was 100% sensitive and 97.7% specific in cadaveric sera; there were no false-negatives.

The potential for transmission of HCV via corneal transplantation stems from many reports of HCV developing in solid organ (e.g., heart, liver, kidney, bone) recipients from HCV-seropositive donors. 9–11 In fact, HCV has been the most prevalent cause of chronic hepatitis in both blood and organ recipients. 9 However, the direct evidence of the presence of HCV in corneas is lacking. It has been shown that only 20–26% of seropositive cornea donors have viral RNA in their serum and, of these, none had viral RNA in the cornea. 8,12 Six recipients of corneas from HCV-seropositive donors, at least two of whom had viral RNA in their serum, did not sero-convert after surgery. 13 It was hypothesized that although infected blood may be transferred within the vascular solid organ or tissue, the human cornea is unlikely to carry a substantial viral load, even if obtained from a viremic donor because of the relative avascular nature of the cornea.

We set out to investigate the correlation between HCV seropositivity and the presence of HCV RNA in such corneas and to provide further (or point out the lack of) evidence to the possible transmissibility of HCV via corneal grafts.



All potential cornea donors are serologically screened for human immunodeficiency virus, hepatitis B virus, and HCV by the Eye Bank of Canada (Ontario Division) in Toronto. Serum samples are collected from all potential donor cadavers and are subsequently kept refrigerated until screened. During a period of 1 year, between April 1995 and March 1996, 15 potential donors tested HCV-positive using the second-generation Abbott HCV EIA 2.0 assay. Their sera were further tested with RIBA-II. In accordance with the EBAA guidelines, the corneas of these donors were not released for surgical use; they were instead processed in our study for identification of HCV RNA.

HCV RNA Extraction

This was done by homogenizing frozen corneal tissue with RNA STAT-50 LS solution (ISO-TEX Diagnostics, Friendswood, TX, U.S.A.). The RNA was then extracted with phenolchloroform and precipitated with ethanol and, finally, resuspended in 5 mL RNASE-free water.

HCV RNA Amplification and Detection by Polymerase Chain Reaction

For detection of HCV RNA, we used the AMPLICOR HCV test (version 2.0; Roche Diagnostics, Branchburg, NJ, U.S.A.), which is a direct DNA probe test that uses a nucleic acid amplification technology called polymerase chain reaction (PCR), as well as nucleic acid hybridization, for the detection of HCV RNA. It is based on four major processes: reverse transcription of target RNA to generate a complementary (c)DNA, PCR target amplification, hybridization of the amplified product to a specific oligonucleotide probe, and detection of the probe-bound amplified product by color formation. Selection of a target RNA sequence depends on identification of regions within the HCV genome that show maximum sequence conservation among the various genotypes. Accordingly, appropriate selection of primers is critical to the ability to detect all of these gene families. The 5-untranslated region of the HCV genome has been shown to have maximum conservation of RNA sequence. The AMPLICOR HCV test uses the primers KY78 and KY80 to define a 244-nucleotide sequence within the highly conserved 5-untranslated region.

Positive and Negative Controls

Both the positive and negative controls are included in the AMPLICOR test kit. One replicate of the AMPLICOR HCV-negative (−) control and one replicate of the AMPLICOR HCV-positive (+) control can be processed with each batch of specimens. Each separate specimen batch can be validated individually by the set of controls included with the batch.

Interpretation of the Test Result

The presence of HCV RNA in a specimen is determined by comparing the absorbance at 450 nm (A450) of the unknown specimen to that of the cutoff value. Specimens with results >0.60 A450 should be interpreted as positive for the presence of HCV RNA. Specimens with results <0.25 A450 should be interpreted as negative. Any specimen within the range of 0.25–0.60 A450 must be reassayed by repeating the specimen processing and is considered positive only if the repeated assay result is >0.60 A450.

Control Acceptance Criteria

The assay result for the AMPLICOR HCV− control should be <0.25 A450. If the AMPLICOR HCV− control is ≥0.25 A450, the run should be invalidated and the entire test procedure (specimen preparation, amplification and detection) should be repeated.

The assay result for the AMPLICOR HCV+ control should be >0.60 A450. If the AMPLICOR HCV+ control is ≤0.60 A450, the run should be invalidated and the entire test procedure (specimen preparation, amplification, and detection) should be repeated.

Further Control from Corneas of Seronegative Donors

As a further sensitivity control, 12 corneas from six seronegative donors were obtained from the Eye Bank of Canada, processed in the exact manner, and then subjected to similar AMPLICOR HCV test for HCV RNA.


The Eye Bank of Canada (Ontario Division) received a total of 3,238 corneas from 1,619 potential donors from April 1995 to March 1996, of which 30 corneas (0.93%) from 15 donors were rejected on the grounds of HCV seropositivity. The mean age of these seropositive donors was 51.7 years (range, 21–74 years).

The RIBA-II confirmatory test was performed only on the cadaveric sera of 10 of these 15 potential donors, as sera of the other 5 were of insufficient quantity for testing. Of the ten tested, seven were confirmed positive by RIBA-II. Therefore, using RIBA-II as the reference standard, the second-generation EIA 2.0 gave three false-positives (3/10 or 30%) (Table 1).

Seropositive donors

Only 29 of the 30 corneas were processed for PCR detection of HCV RNA as 1 cornea was inadvertently misplaced. HCV RNA was detected in 7 of the 29 corneas (24.1%). (Table 1). If we consider only those corneas from donors with positive RIBA-II, then HCV RNA was found in 4 out of 13 (30.8%) tested corneas. As a further sensitivity test, HCV RNA was detected in none of the 12 corneas from the six seronegative controls.


HCV is a single-stranded RNA virus, with a genome of approximately 10,000 nucleotides coding for 3,000 amino acids. Serologic testing for HCV in conjunction with epidemiologic studies have verified that HCV is the major cause of parenterally transmitted non-A, non-B hepatitis. 14 HCV affects 1–2% of the population in the United States, 15 and our study shows almost similar trends, i.e., about 1% of cornea donors were HCV-seropositive. Although 25% of patients with acute hepatitis have no sequelae, the rest may develop chronic infection with an increased risk of liver cirrhosis (20–50%). 15 Association between chronic HCV infection and hepatocellular carcinoma has also been established. Therefore, the potential of transmissibility and the serious clinical consequences of HCV infection warrant a careful screening of potential seropositive donors.

Our study demonstrated for the first time the presence of HCV RNA in corneal tissues obtained from HCV EIA 2.0-seropositive donors, by using the AMPLICOR HCV test. We detected HCV RNA in 10 out of 29 (34.5%) corneas from HCV EIA 2.0-seropositive donors. Even if we consider only those corneas from donors who tested positive with both HCV EIA 2.0 and RIBA II, there was still a significant proportion of the corneas (4/13 or 30.8%) with HCV RNA. It is noteworthy that two pairs of corneas had one PCR-positive cornea whose mate was negative.

Previous studies have shown no correlation between HCV seropositivity and the presence of HCV RNA in the corneal tissues. 3,12 Krajden et al. 16 further demonstrated the transmission of HCV to five solid organ transplant recipients but not to the two corneal transplant recipients, although all the organs and tissues were from the same HCV-seropositive donor. They suggested that this may be a reflection of a low viral load in corneal tissue due to its avascular nature, loss of virus after washing, and preservation technique, or a low risk of infection after ocular exposure. The absence of documented transmission of disease, coupled with the lack of correlation between HCV seropositivity and presence of HCV RNA in cornea, has led some authors to question the value of serologic testing for HCV and the justification of rejecting donor corneas on the grounds of donor HCV seropositivity. In this respect, our study reinforces the rationale of Food and Drug Administration and EBAA guidelines and helps settle the controversy surrounding the potential transmissibility of HCV via corneal transplantation.

Currently, there are many commercially available tests that use the PCR technique for the detection of HCV RNA, and the AMPLICOR HCV test that we used in our study has been shown to be one of the most sensitive and specific ones. 17 This test also has no cross-reactivity with other viruses, such as hepatitis A virus, hepatitis B virus, human immunodeficiency virus, or cytomegalovirus. However, it must be mentioned that one of the procedural limitations of this test is that it has been validated for use with only human serum or human plasma collected in ethylenediaminetetraacetic acid or acid citrate dextrose solution anticoagulants. Testing of other specimen types, therefore, may result in false-negative or -positive results.

Although the detection of nucleic acid does not necessarily correlate with the presence of a replication competent virus, it is clear that under certain circumstances free nucleic acid replicons can cause active infection. 18 Thus, although the risk of transmission from corneas is low, the potential for transmission exists. Nucleic acid testing has become the standard of care for blood product testing and will be the standard for organ testing when turnaround times improve.

One of the problems of EIA testing for HCV antibodies is that the test does not differentiate between past and current HCV infection because antibodies may still be present several months after the infection has cleared. Furthermore, it has a relatively high rate of false-positives, especially in low-prevalence populations (e.g., the United States). 15 Laycock et al. 12 showed that second-generation EIA had a specificity of 92.7% and a sensitivity of 100%. Our data showed a much higher percentage of false-positives, in the region of 30%. This may lead to a waste of otherwise transplantable corneal tissues based on the Food and Drug Administration's guideline that prohibits surgical use of cornea from donors who screen HCV-positive regardless of confirmatory test results. Nonetheless, we believe that such guidelines should be upheld as it is better to err on the safe side in view of the possible grave clinical sequelae of HCV infection.

Another problem of screening is the possibility of not detecting the rare seronegative but still infected donors, namely those in the early window before seroconversion (82 days for HCV) 19 or those with false-negative serology. Fortunately, to date, no such donors have been documented to transmit HCV hepatitis. Nevertheless, a possible explanation could be that HCV might be difficult or impossible to transmit via corneal transplantation.

In conclusion, our study demonstrated for the first time a significant correlation between HCV seropositivity and the presence of HCV in corneas. Routine HCV serologic testing for all potential cornea donor and rejection of corneal tissues based on HCV seropositivity are certainly justifiable in view of the potential transmissibility and the grave clinical consequence of HCV infection.


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Hepatitis C virus; HCV-seropositive cornea donors; Polymerase chain reaction; Immunoassay; Screening

© 2001 Lippincott Williams & Wilkins, Inc.