Repurposing of the antibiotic nitroxoline for the treatment of mpox.

The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.

Identification of Natural Products Inhibiting SARS-CoV-2 by Targeting Viral Proteases: A Combined in Silico and in Vitro Approach.

In this study, an integrated in silico-in vitro approach was employed to discover natural products (NPs) active against SARS-CoV-2. The two SARS-CoV-2 viral proteases, i.e., main protease (Mpro) and papain-like protease (PLpro), were selected as targets for the in silico study. Virtual hits were obtained by docking more than 140,000 NPs and NP derivatives available in-house and from commercial sources, and 38 virtual hits were experimentally validated in vitro using two enzyme-based assays. Five inhibited the enzyme activity of SARS-CoV-2 Mpro by more than 60% at a concentration of 20 µM, and four of them with high potency (IC50 < 10 µM). These hit compounds were further evaluated for their antiviral activity against SARS-CoV-2 in Calu-3 cells. The results from the cell-based assay revealed three mulberry Diels-Alder-type adducts (MDAAs) from Morus alba with pronounced anti-SARS-CoV-2 activities. Sanggenons C (12), O (13), and G (15) showed IC50 values of 4.6, 8.0, and 7.6 µM and selectivity index values of 5.1, 3.1 and 6.5, respectively. The docking poses of MDAAs in SARS-CoV-2 Mpro proposed a butterfly-shaped binding conformation, which was supported by the results of saturation transfer difference NMR experiments and competitive 1H relaxation dispersion NMR spectroscopy.

Aerosol measurement identifies SARS-CoV 2 PCR positive adults compared with healthy controls.

BACKGROUND: SARS-CoV-2 is spread primarily through droplets and aerosols. Exhaled aerosols are generated in the upper airways through shear stress and in the lung periphery by 'reopening of collapsed airways'. Aerosol measuring may detect highly contagious individuals ("super spreaders or super-emitters") and discriminate between SARS-CoV-2 infected and non-infected individuals. This is the first study comparing exhaled aerosols in SARS-CoV-2 infected individuals and healthy controls. DESIGN: A prospective observational cohort study in 288 adults, comprising 64 patients testing positive by SARS CoV-2 PCR before enrollment, and 224 healthy adults testing negative (matched control sample) at the University Hospital Frankfurt, Germany, from February to June 2021. Study objective was to evaluate the concentration of exhaled aerosols during physiologic breathing in SARS-CoV-2 PCR-positive and -negative subjects. Secondary outcome measures included correlation of aerosol concentration to SARS-CoV-2 PCR results, change in aerosol concentration due to confounders, and correlation between clinical symptoms and aerosol. RESULTS: There was a highly significant difference in respiratory aerosol concentrations between SARS-CoV-2 PCR-positive (median 1490.5/L) and -negative subjects (median 252.0/L; p < 0.0001). There were no significant differences due to age, sex, smoking status, or body mass index. ROC analysis showed an AUC of 0.8918. CONCLUSIONS: Measurements of respiratory aerosols were significantly elevated in SARS-CoV-2 positive individuals, which helps to understand the spread and course of respiratory viral infections, as well as the detection of highly infectious individuals.

[Evaluation of Characteristics and Management of Influenza Patients in Relation to the STIKO Vaccination Recommendation During the 2017/2018 Season]. / Evaluation von Charakteristika und Management von Influenzapatienten in Bezug auf die STIKO Impfempfehlung während der Saison 2017/2018.

BACKGROUND: Every year a large number of patients is suffering from influenza infection with often severe outcome. The influenza season 2017/2018 was characterized by a high number of cases (in Germany>346,000 laboratory-confirmed cases), but also by a high rate of hospitalizations with sometimes severe clinical outcome - also in the group of patients under 60 years. AIM: The aim of the present study was to find out whether patients not fullfilling the STIKO vaccination recommendation in the 2017/18 season were suffering from a worse outcome. MATERIALS AND METHODS: All laboratory-confirmed influenza patients at Frankfurt University Hospital were retrospectively analyzed for disease severity with respect to the primary endpoint. Secondary endpoints were defined as demographic data, length of hospital stay, previous illnesses, intensive care therapy and its duration, drug therapy, and mortality. RESULTS: Fifty-one of 303 patients (16.8%) required intensive care treatments. Of these 51, 46 patients (90.2%) belonged to the group that should have been vaccinated according to the vaccination recommendations according to STIKO, 5 patients (9.8%) did not belong to this group (p=0.434). Of the 51 ICU patients, 16 (31.4%) died. All deceased were from the group with vaccination recommendation (p=0.120). CONCLUSIONS: Based on these data, it appears that severe disease progression occurs in both the group of patients with and without STIKO vaccination recommendation, but deaths occur only in the group of patients with recommendation.

Heterologous immunization with BNT162b2 followed by mRNA-1273 in dialysis patients: seroconversion and presence of neutralizing antibodies.

INTRODUCTION: The vital renal replacement therapy makes it impossible for dialysis patients to distance themselves socially. This results in a high risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and developing coronavuris disease 2019, with excess mortality due to disease burden and immunosuppression. We determined the efficacy of a 100-µg booster of mRNA-1273 (Moderna, Cambridge, MA, USA) 6 months after two doses of BNT162b2 (BioNTech/Pfizer, Mainz, Germany/New York, USA) in 194 SARS-CoV-2-naïve dialysis patients. METHODS: Anti-SARS-CoV-2 spike antibodies were measured with the Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics, Mannheim, Germany) 4 and 10-12 weeks after two doses of BNT162b2 as well as 4 weeks after the mRNA-1273 booster. The presence of neutralizing antibodies was measured by the SARS-CoV-2 Surrogate Virus Neutralization Test (GenScript Biotech, Piscataway, NJ, USA). Two different cut-offs for positivity were used, one according to the manufacturer's specifications and one correlating with positivity in a plaque reduction neutralization test (PRNT). Receiver operating characteristics analyses were performed to match the anti-SARS-CoV-2 spike antibody cut-offs with the cut-offs in the surrogate neutralization assay accordingly. RESULTS: Any level of immunoreactivity determined by the anti-SARS-CoV-2 spike antibody assay was found in 87.3% (n = 144/165) and 90.6% (n = 164/181) of patients 4 and 10-12 weeks, respectively, after two doses of BNT162b2. This was reduced to 68.5% or 60.6% 4 weeks and 51.7% or 35.4% 10-12 weeks, respectively, when using the ROC cut-offs for neutralizing antibodies in the surrogate neutralization test (manufacturer's cut-off ≥103 U/mL and cut-off correlating with PRNT ≥196 U/mL). Four weeks after the mRNA-1273 booster, the concentration of anti-SARS-CoV-2 spike antibodies increased to 23 119.9 U/mL and to 97.3% for both cut-offs of neutralizing antibodies. CONCLUSION: Two doses of BNT162b2 followed by one dose of mRNA-1273 within 6 months in patients receiving maintenance dialysis resulted in significant titres of SARS-CoV-2 spike antibodies. While two doses of mRNA vaccine achieved adequate humoral immunity in a minority, the third vaccination boosts the development of virus-neutralizing quantities of SARS-CoV-2 spike antibodies (against wild-type SARS-CoV-2) in almost all patients.

Suitability of Different Diagnostic Platforms for Virological Testing of Blood Samples from Cornea Donors.

Background: To minimize the risk of disease transmission in cornea transplantation, donor screening for blood-derived viral infections is mandatory. Ideally, pre-mortem blood samples are used, but based on availability, cadaveric blood samples of cornea donors may also be used. However, serological and nucleic acid amplification tests (NATs) need to be validated for the use of cadaveric specimens. Methods: Hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV) 1/2, and Treponema pallidum (syphilis)-specific serological and/or NAT assays were validated on different platforms (Abbott Alinity i, Alinity m, Roche Cobas 6800, and Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM)) using (un)spiked paired pre- and post-mortem cornea donor blood samples from the same individual (up to 23.83 h after death) of 28 individuals in accordance with the specifications of the German Federal Institute for Vaccines and Biomedicines (Paul-Ehrlich-Institut [PEI]). In addition, routinely HBV-, HCV- and HIV-PCR-negative tested post-mortem blood samples of 24 individuals were used to assess NAT specificity. Results: For the majority of serological parameters on the Abbott Alinity i (HBsAg, anti-HBc, anti-HBs, anti-HCV, anti-HIV, anti-HTLV 1/2, and anti-Treponema pallidum), ratios of generated test results of (un)spiked paired pre- and post-mortem blood samples differed ≤25%, with an agreement of qualitative pre- and post-mortem test results ranging from 91.2 to 100%. For NAT parameters (HBV, HCV, and HIV) on the Cobas 6800, Alinity m, and CAP/CTM, no significant deviation in virus concentrations (factor >5) of spiked pre- and post-mortem blood samples could be observed. Ct-values of corresponding internal controls did also not differ significantly (>1.5 Ct-values). In addition, no false-positive test results were generated when specificity was assessed. Conclusion: Overall, fluctuations of test results for serological and NAT parameters in pre- and post-mortem blood samples examined in this study, were only limited and within the range of what is also observed when routinely testing fresh patient specimens. We conclude that all examined assays are eligible for the screening of blood samples taken up to about 24 h after the occurrence of death.

[Disinfectants during the COVID-19 pandemic: a challenge]. / Desinfektionsmittel in der COVID-19-Pandemie: eine Herausforderung.

Disinfection measures have become more important as a result of the COVID-19 pandemic in Germany. The increased need for disinfectants at the beginning of the pandemic required temporary legal regulations in order to provide a sufficient quantity of products for the necessary disinfection in the medical sector on the one hand and for the additional demand in the population on the other. For this purpose, the Federal Institute for Drugs and Medical Devices (BfArM) and the Federal Institute for Occupational Safety and Health (BAuA) issued a general ruling, which is explained in more detail in this article. The focus was on measures for hygienic hand disinfection. However, other applications such as surface disinfection in relation to pandemic respiratory diseases are also addressed. The experience gained in ensuring the supply of disinfectants that are effective and safe to use should be used to prepare for further pandemics.

Limited Neutralization of Authentic Severe Acute Respiratory Syndrome Coronavirus 2 Variants Carrying E484K In Vitro.

Whether monoclonal antibodies are able to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has been investigated using pseudoviruses. In this study we show that bamlanivimab, casirivimab, and imdevimab efficiently neutralize authentic SARS-CoV-2, including variant B.1.1.7 (alpha), but variants B.1.351 (beta) and P.2 (zeta) were resistant against bamlanivimab and partially resistant to casirivimab. Whether antibodies are able to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variantshas been investigated using pseudoviruses. We show that authentic SARS-CoV-2 carrying E484K were resistant against bamlanivimab and less susceptible to casirivimab, convalescent and vaccine-elicited sera.

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings.

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.

Retrospectively diagnosing congenital cytomegalovirus infections in symptomatic infants is challenging.

AIM: Our aim was to analyse the diagnostic workup of hospitalised infants with symptoms of congenital cytomegalovirus (CMV) infections. METHODS: This retrospective study was carried out at the University Hospital Frankfurt, Germany, from 2008 to 2017 on infants aged 4 weeks to 12 months presenting with neurological symptoms consistent with congenital CMV infections. RESULTS: We studied 117 infants, and workup data for CMV infections were available for 84%. Of these, 54% were immunoglobulin G- and immunoglobulin M-seronegative for CMV or immunoglobulin G-seropositive with no viral shedding. Congenital CMV infection was excluded in these cases. In 16%, the CMV workup was incomplete, precluding a definitive diagnosis. Dried blood spots (DBS) were requested from 30%. CMV polymerase chain reaction was negative in 19 of these 29 infants, and CMV deoxyribonucleic acid detection confirmed congenital CMV infections in six patients. DBS had been destroyed in line with German law in four cases. Congenital CMV infections were diagnosed (5%) or excluded (62%) in 67% of patients and unanswered in the remaining 33%. CONCLUSION: Diagnoses of congenital CMV infections were widely considered and found in 5%. CMV was not stringently investigated in all patients or remained elusive due to German law on destroying DBS.

Typical symptoms of common otorhinolaryngological diseases may mask a SARS-CoV-2 infection.

PURPOSE: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) replicates predominantly in the upper respiratory tract and is primarily transmitted by droplets and aerosols. Taking the medical history for typical COVID-19 symptoms and PCR-based SARS-CoV-2 testing have become established as screening procedures. The aim of this work was to describe the clinical appearance of SARS-CoV-2-PCR positive patients and to determine the SARS-CoV-2 contact risk for health care workers (HCW). METHODS: The retrospective study included n = 2283 SARS-CoV-2 PCR tests from n = 1725 patients with otorhinolaryngological (ORL) diseases performed from March to November 2020 prior to inpatient treatment. In addition, demographic data and medical history were assessed. RESULTS: n = 13 PCR tests (0.6%) were positive for SARS-CoV-2 RNA. The positive rate showed a significant increase during the observation period (p < 0.01). None of the patients had clinical symptoms that led to a suspected diagnosis of COVID-19 before PCR testing. The patients were either asymptomatic (n = 4) or had symptoms that were interpreted as symptoms typical of the ORL disease or secondary diagnoses (n = 9). CONCLUSION: The identification of SARS-CoV-2-positive patients is a considerable challenge in clinical practice. Our findings illustrate that taking a medical history alone is of limited value and cannot replace molecular SARS-CoV-2 testing, especially for patients with ORL diseases. Our data also demonstrate that there is a high probability of contact with SARS-CoV-2-positive patients in everyday clinical practice, so that the use of personal protective equipment, even in apparently "routine cases", is highly recommended.

Algorithms for the Testing of Tissue Donors for Human Immunodeficiency Virus, Hepatitis B Virus, and Hepatitis C Virus.

BACKGROUND: Although transmission of pathogenic viruses through human tissue grafts is rare, it is still one of the most serious dreaded risks of transplantation. Therefore, in addition to the detailed medical and social history, a comprehensive serologic and molecular screening of the tissue donors for relevant viral markers for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) is necessary. In the case of reactive results in particular, clear decisions regarding follow-up testing and the criteria for tissue release must be made. METHODS: Based on the clinical relevance of the specific virus markers, the sensitivity of the serological and molecular biological methods used and the application of inactivation methods, algorithms for tissue release are suggested. RESULTS: Compliance with the preanalytical requirements and assessment of a possible hemodilution are mandatory requirements before testing the blood samples. While HIV testing follows defined algorithms, the procedures for HBV and HCV diagnostics are under discussion. Screening and decisions for HBV are often not as simple, e.g., due to cases of occult HBV infection, false-positive anti-HBc results, or early window period positive HBV NAT results. In the case of HCV diagnostics, modern therapies with direct-acting antivirals, which are often associated with successful treatment of the infection, should be included in the decision. CONCLUSION: In HBV and HCV testing, a high-sensitivity virus genome test should play a central role in diagnostics, especially in the case of equivocal serology, and it should be the basis for the decision to release the tissue. The proposed test algorithms and decisions are also based on current European recommendations and standards for safety and quality assurance in tissue and cell banking.

Clinical performance of different SARS-CoV-2 IgG antibody tests.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays are urgently needed for rapid diagnosis, contact tracing, and for epidemiological studies. So far, there is limited data on how commercially available tests perform with real patient samples, and if positive tested samples show neutralizing abilities. Focusing on IgG antibodies, we demonstrate the performance of two enzyme-linked immunosorbent assay (ELISA) assays (Euroimmun SARS-CoV-2 IgG and Vircell COVID-19 ELISA IgG) in comparison to one lateral flow assay (FaStep COVID-19 IgG/IgM Rapid Test Device) and two in-house developed assays (immunofluorescence assay [IFA] and plaque reduction neutralization test [PRNT]). We tested follow up serum/plasma samples of individuals polymerase chain reaction-diagnosed with COVID-19. Most of the SARS-CoV-2 samples were from individuals with moderate to the severe clinical course, who required an in-patient hospital stay. For all examined assays, the sensitivity ranged from 58.8 to 76.5% for the early phase of infection (days 5-9) and from 93.8% to 100% for the later period (days 10-18).

SARS-CoV-2 asymptomatic and symptomatic patients and risk for transfusion transmission.

Oral swabs, sputum, and blood samples from 18 asymptomatic and symptomatic patients with SARS-CoV-2 infection were examined using RT-PCR testing in order to assess the risk of transfusion-related transmission. In asymptomatic patients as well as patients with flu-like symptoms and fever, no SARS-CoV-2 RNA could be detected in the blood or serum despite a clearly positive result in all throat swabs. As patients with symptoms of infectious disease will not be admitted to blood donation, the risk for transfusion transmission of SARS-CoV-2 seems to be negligible.

Comparative multi-assay evaluation of Determine™ HIV-1/2 Ag/Ab Combo rapid diagnostic tests in acute and chronic HIV infection.

In resource-limited or point-of-care settings, rapid diagnostic tests (RDTs), that aim to simultaneously detect HIV antibodies and p24 capsid (p24CA) antigen with high sensitivity, can pose important alternatives to screen for early infections. We evaluated the performance of the antibody and antigen components of the old and novel version of the Determine™ HIV-1/2 Ag/Ab Combo RDTs in parallel to quantifications in a fourth-generation antigen/antibody immunoassay (4G-EIA), p24CA antigen immunoassay (p24CA-EIA), immunoblots, and nucleic acid quantification. We included plasma samples of acute, treatment-naïve HIV-1 infections (Fiebig stages I-VI, subtypes A1, B, C, F, CRF02_AG, CRF02_AE, URF) or chronic HIV-1 and HIV-2 infections. The tests' antigen component was evaluated also for a panel of subtype B HIV-1 transmitted/founder (T/F) viruses, HIV-2 strains and HIV-2 primary isolates. Furthermore, we assessed the analytical sensitivity of the RDTs to detect p24CA using a highly purified HIV-1NL4-3 p24CA standard. We found that 77% of plasma samples from acutely infected, immunoblot-negative HIV-1 patients in Fiebig stages II-III were identified by the new RDT, while only 25% scored positive in the old RDT. Both RDTs reacted to all samples from chronically HIV-1-infected and acutely HIV-1-infected patients with positive immunoblots. All specimens from chronically infected HIV-2 patients scored positive in the new RDT. Of note, the sensitivity of the RDTs to detect recombinant p24CA from a subtype B virus ranged between 50 and 200 pg/mL, mirrored also by the detection of HIV-1 T/F viruses only at antigen concentrations tenfold higher than suggested by the manufacturer. The RTD failed to recognize any of the HIV-2 viruses tested. Our results indicate that the new version of the Determine™ HIV-1/2 Ag/Ab Combo displays an increased sensitivity to detect HIV-1 p24CA-positive, immunoblot-negative plasma samples compared to the precursor version. The sensitivity of 4G-EIA and p24CA-EIA to detect the major structural HIV antigen, and thus to diagnose acute infections prior to seroconversion, is still superior.

Thirty years of CMV seroprevalence-a longitudinal analysis in a German university hospital.

Human cytomegalovirus (CMV) is a significant cause of morbidity and mortality in patient groups at risk. We have previously shown that the anti-CMV IgG seroprevalence in an urban region of Germany has changed over the last decades. Overall, a decline from 63.7 to 57.25% had been observed between 1988-1997 and 1998-2008 (p < 0,001). Here, we continuously follow the trends to the most recent decade 2009 to 2018. In a retrospective analysis, we determined the seroprevalence of CMV IgG antibodies in our patient cohort, stratified by gender and selected groups at risk (e.g., patients with HIV infection; women of childbearing age). The overall prevalence of anti-CMV IgG non-significantly declined further from 57.25% in 1998-2008 to 56.48% in 2009-2018 (p = 0.881). Looking at gender differences, overall CMV seroprevalence in males declined to 52.82% (from 55.54% in 1998-2008; p = 0.0254), while it non-significantly increased in females to 59.80%. The high seroprevalence in patients with a known HIV infection further increased from 87.46% in 1998-2008 to 92.93% in the current period (p = 0.9999). In women of childbearing age, no significant changes over the last three decades could be observed. The CMV seroprevalence in oncological patients was determined to be 60.64%. Overall, the former significant decline of CMV seroprevalence between the decades 1988-1997 and 1998-2008 in this urban region of Germany slowed down to a non-significant decrease of 0.77% (1998-2008 vs. 2009-2018). This might be an indicator that CMV seroprevalence has reached a plateau.

HCV RNA Testing of Plasma Samples from Cornea Donors: Suitability of Plasma Samples Stored at 4 °C for up to 8 Days.

BACKGROUND: The HCV RNA testing of potential cornea donors frequently relies on blood samples stored pre mortem. The recommended storage time of maximum 72 h frequently excludes a significant fraction of donors. METHODS: The influence of storage time of EDTA plasma samples at 4 °C on the viral load measured with the Roche HCV Quantitative Test vs. 2.0 was evaluated for 43 samples from HCV-positive individuals. RESULTS: The mean reduction of the viral load after 4 °C storage for 6-8 days was 0.46 log10 IU/ml (range +0.17 to -1.66 log10 IU/ml). After 1-3 days a mean loss of 0.19 log10 IU/ml (range +0.30 to -1.41 log10 IU/ml) and after 3-5 days of 0.32 log10 IU/ml (range +0.36 to -1.81 log10 IU/ml) was observed. In 23.3% of samples, a viral load reduction ≥ 1 log10 IU/ml (1.0-1.81 log10 IU/ml) was found after prolonged storage (5-8 days). In none of the samples did the HCV load fall below the detection limit. CONCLUSION: Plasma storage for up to 8 days can quantitatively reduce the HCV RNA load, yet has no influence on the reliability of a qualitative HCV RNA detection by this ultrasensitive test to determine the HCV status of serologically negative cornea donors.