EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Our objective was to evaluate the serologic positivity of cornea donors and microbiologic positivity of cornea storage media at the Ege University Tissue and Cornea Bank, Izmir, Turkey.

Materials and Methods: We retrospectively investigated the serologic blood sample and microbiological culture media analysis results of all cornea donors at Ege University Tissue and Cornea Bank between 2007 and 2015 with reference to age, sex, and cause of death of each donor.

Results: Mean age of the 955 deceased donors was 43.19 ± 15.89 years (range, 2-65 y). The mean postmortem time to blood sample removal and excision of the cornea tissue was 8.4 hours (range, 4-12 h). Serologic analyses showed that 855 donors (89.5%) were seronegative. The remaining donors were seropositive for hepatitis B (54 donors; 5.7%), human immunodeficiency (27 donors; 2.8%), hepatitis C (14 donors; 1.5%), and syphilis (5 donors; 0.5%) virus infections. Microbiologic analyses of the storage media were negative, with no microorganisms shown in 855 samples (89.5%). Candida species (32 donors; 3.4%), Escherichia coli (14 donors; 1.5%), Pseudomonas aeruginosa (11 donors; 1.2%), methicillin-resistant Staphylococcus aureus (11 donors; 1.2%), Enterobacter species (11 donors; 1.2%), Klebsiella pneumoniae (7 donors; 0.7%), Acinetobacter baumannii (6 donors; 0.6%), Proteus species (5 donors; 0.5%), and Corynebacterium species (3 donors; 0.3%) were the detected microorganisms in the infected storage media.

Conclusions: False-positive serologic results among cornea donors were high. The incidence of false-positive results might be decreased by earlier blood removal from deceased donors and testing of all potential donors in intensive care units. Although rare, endophthalmitis after keratoplasty might be a devastating problem. In addition to serologic testing, microbiologic analyses of cornea storage media before transplant may be an effective way to prevent postoperative infectious complications.

Key words : Keratoplasty, Microbiology, Serology

Tissues from deceased donors, ranging from musculoskeletal tissue to cornea and cardiovascular allografts, are increasingly being used in many clinical fields. Postmortem tissue donation has become an indispensable basis for therapeutically necessary allogenic tissue transplant. By law, defined serologic examinations of deceased cornea donor’s blood are required to minimize the risk of transmission of infectious diseases to the recipient of a tissue graft. When blood samples of the donor before death are not available, postmortem samples can be used for serologic testing. However, these postmortem samples must not be taken later than 24 hours after excitus.¹

Although sterility steps are rigidly followed, corneas from deceased donors may still become contaminated during explantation or processing. Postoperative infectious keratitis and endophthalmitis are the most severe transplant-related complications after corneal transplant and may have devastating effects.^2-4 Thorough donor screening, decontamination of donor tissue, and microbiologic screening of culture media are priorities of an eye bank aiming to reduce the risk of corneal graft infection. Moreover, these measures may help to minimize the number of discarded corneas as a result of contamination.^5,6 To identify these contaminated corneas, the storage media also requires microbiologic analyses.⁷

The aim of the present study was to evaluate the serologic positivity of cornea donors and microbiologic positivity of cornea storage media at the Ege University Tissue and Cornea Bank, Izmir, Turkey.

Materials and Methods

We retrospectively examined the serologic blood sample and microbiologic culture medium analysis results of all corneas donated to Ege University Tissue and Cornea Bank between May 1, 2007, and November 15, 2015, with reference to age, sex, and cause of death of each donor.

Serologic analyses
To minimize the risk of transmission of any infection through corneal transplant, special attention is given to tests of infectious diseases, including hepatitis B surface antigen for hepatitis B, anti-hepatitis C virus for hepatitis C, anti-human immunodeficiency virus 1 and 2 for human immunodeficiency virus 1 and 2, and trepanoma pallidum immunoglobulin M and immunoglobulin G for syphilis. All of these tests were performed with the ARCHITECT i1000SR Immunoassay Analyzer (Abbott, Wiesbaden, Germany). The tissue was considered as unsuitable with a positive screening test.

Microbiologic analyses of the cornea storage medium
For preservation, corneas were placed in cornea storage medium (Optisol GS, Chiron Intraoptics, Irvine, CA, USA) and left at room temperature for 12 to 18 hours. One milliliter of the liquid medium was subsequently removed under sterile conditions and inoculated into blood culture bottles of a commercially available bacterial detection system (BacT/ALERT 3D automated system, bio Mérieux, Durham, NC, USA) and sent to our bacteriology laboratory. The culture bottles were incubated in the BacT/ALERT 3D system for 7 days. Microorganisms from culture-positive bottles were identified by using conventional biochemical procedures and an automated bacterial identification system (VITEK 2, bio Mérieux, Marcy-l’Etoile, France).

Mean age of the 955 deceased donors was 43.19 ± 15.89 years (range, 2-65 y). Male-to-female ratio was 680:275 (71.2% men vs 28.8% women). Causes of death were cardiopulmonary arrest (365 donors; 38.2%), respiratory dysfunction (162 donors; 17.0%), traffic accident (149 donors; 15.6%), subarachnoid bleeding (75 donors; 7.9%), cerebral hemorrhage (64 donors; 6.7%), fall from height (50 donors; 5.2%), firearm injury (40 donors; 4.2%), head trauma (16 donors; 1.7%), suicide (11 donors; 1.5%), intoxication (6 donors; 0.6%), drowning (6 donors; 0.6%), and electric injury (8 donors; 0.8%).

The mean postmortem time to blood sample removal and postmortem time of excision of the cornea tissue was 8.4 hours (range, 4-12 h).

The serologic analyses showed that 855 donors (89.5%) were seronegative. The remaining donors were seropositive for hepatitis B virus (54 donors; 5.7%), human immunodeficiency virus (27 donors; 2.8%), hepatitis C virus (14 donors; 1.5%), and syphilis (5 donors; 0.5%). All seropositive donor tissues (100 donors; 10.5%) were destroyed.

The microbiologic analysis of the storage media was negative, with no microorganism shown in 855 samples (89.5%). The remaining samples were positive, with cultured microorganisms as follows; Candida species (32 donors; 3.4%), Escherichia coli (14 donors; 1.5%), Pseudomonas aeruginosa (11 donors; 1.2%), methicillin-resistant Staphylococcus aureus (11 donors; 1.2%), Enterobacter species (11 donors; 1.2%), Klebsiella pneumoniae (7 donors; 0.7%), Acinetobacter baumannii (6 donors; 0.6%), Proteus species (5 donors; 0.5%), and Corynebacterium species (3 donors; 0.3%).

Defined serologic blood tests of deceased cornea donors are required to minimize the risk of possible infectious transmission to transplant recipients.¹ False-negative serology results of postmortem blood samples occur in less than 1% of donations.¹ However, false-positive results are much more common, at about 3.5%.¹ It is known that hemolysis, autolysis, and bacterial contamination may produce significant changes to postmortem blood samples, which could lead to false serologic test results.1 Challine and associates8 examined 565 consecutive deceased donors for serologic markers of human immunodeficiency virus types 1 and 2, human T-cell leukemia virus type 1, and hepatitis B and hepatitis C viruses. In 19.1% of the samples, positive results were detected; these were rejected because confirmation testing showed unequivocal results. No false-negative results occurred in their study. Wilkemeyer and associates1 reported on 487 cornea donors and found that 4.3% of donations showed discrepancies between serologic test results from pre- and postmortem blood samples. False-negative results within postmortem serology occurred in 0.8% of cases, and false-positive results within postmortem blood samples occurred at a much more frequent rate of 3.5%. In our study, the seropositivity rate was 10.5%, with all seropositive cornea tissues destroyed.

Challine and associates⁸ stated that there seems to be a tendency of increased false-positive results with increasing postmortem time. Data from Wilkemeyer and associates¹ also support this conclusion. However, a cut-off for the age of postmortem blood samples cannot be clearly determined. Differences in handling of the postmortem blood samples and delayed centrifugation could be important causes for false-positive test results. In our study, the postmortem time to blood sample removal was 4 to 12 hours.

Up to now, there are no published data showing that preprocessing microbiologic testing has a predictive value for postkeratoplasty infections; however, testing is routinely performed for cornea storage medium culture for every single cornea tissue in our eye bank. After preservation of the cornea, we allow cornea storage medium to sit at room temperature for 12 to 18 hours to enhance the efficiency of the in vivo applied antibiotics (streptomycin and gentamicin) in the Optisol-GS solution and to induce microbiologic proliferation if any contamination is positive.^9,10 Later, under sterile conditions, we remove 1 mL of the liquid medium, which is then inoculated into blood culture bottles and sent for microbiologic examination. It is noteworthy that blood culture bottles have been shown to be more sensitive and allow more rapid detection of microbial contamination than conventional microbiologic methods, enabling the earlier release of donor corneas without compromising microbiologic safety.¹¹ The microbiologic testing of the corneoscleral rim can also be performed before storage, with the organ culture medium then tested before transplant. However, because we do not have enough corneal tissue to necessitate long-term storage, it is more practical to test the corneal storage medium. It is suspected that the antimicrobials (streptomycin, penicillin, and amphotericin B13) in the organ culture medium might reduce the sensitivity for microorganism isolation. Therefore, it is reasonable to consider the use of culture systems that contain antibiotic-absorbing resins that inactivate the antibiotics in the organ culture medium to enhance the sensitivity of microbial growth.¹² In our laboratory, the culture system and the blood culture bottles also work on this principle.Incidence of endophthalmitis after keratoplasty varies from 0.08% to 0.77%.13-15 Cause of death is one of the reported factors to be associated with endophthalmitis.15 Donors who die from infection are shown to be related to postkeratoplasty endophthalmitis.¹³ Moreover, donors who were in intensive care units for long duration before death are more likely to transmit nosocomial infections. To avoid undesired complications, we never receive corneal tissue from infection-positive donors. In our clinic from 2007 to 2015, 846 keratoplasty procedures have been performed. Among these keratoplasty recipients, only 1 patient (0.11%) developed corneal graft-related endophthalmitis due to Klebsiella pneumonia.⁴ In this case, microbiologic analysis of the transplanted cornea storage medium was negative. The surveillance cultures (rectal and oropharyngeal swabs) taken from the patient and the operation room also did not yield any similar pathogen. It can be speculated that postoperative contamination probably caused the infection. Our group did not encounter any further extended spectrum beta-lactamase-producing Klebsiella pneumoniae-related nosocomial infections in our clinic. For this reason, this case was considered to be a sporadic infection.

The low rate of endophthalmitis after keratoplasty at our eye bank might be related to the microbiologic analysis of the corneal storage media before transplant of the cornea.

Natural changes that occur in blood after death may result in false-positive results in antigen and antibody detection tests, which are performed to identify markers of viral infection in potential cornea donors. The false-positive serologic results among the cornea donors were found to be high in our series, as mentioned previously.^1,6 False-positive results could be decreased by earlier blood removal from the deceased donors and by testing all potential donors in intensive care units while their hearts are still beating, although this may not be always doable. Although rare, endophthalmitis after keratoplasty is a devastating problem. In addition to serologic testing, microbiologic analyses of the cornea storage media before transplant could be an effective way to prevent postoperative infections.

Volume: 15 Issue: 6 December 2017

FULL TEXT