title: Depletion of hepatitis C virus from procured kidneys using pulsatile perfusion preservation.
abstract: The safety of the use of kidneys procured from a hepatitis C virus (HCV)-positive cadaver donors in transplantation has recently been the subject of controversy. One factor that is important in determining the transmission of the virus and/or viral liver disease is the total viral inoculum to which the renal allograft recipient is exposed as a result of the transplant. We have studied the effect of a standard pulsatile renal preservation procedure and variations of it on the number of viral copies in organs from HCV-positive donors. An HCV-RNA quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was utilized with a recombinant competitive inhibitor substrate added after cDNA synthesis with the PCR primers within the relatively non-polymorphic 5' untranslated region of the HCV genome. Additionally, strain specificity was found to be detectable using a modification of the technique of restriction fragment-length polymorphism (RFLP-PCR), so that virus from the organ donor could be specifically identified and quantified in the recipient. It was observed that standard preservation procedures using pulsatile perfusion were able to eliminate 75% of the virus from the organ in 20 hr. By modifying this procedure to include additional wash steps and a second pulsatile perfusion, greater than 99% of the virus could be eliminated from the kidney. In a related study, we used quantitative PCR to study requirements for filtration of the virus using HCV-positive serum. It was found that a high-flow-rate ultrafilter with a molecular weight cut-off (MWCO) of 300,000 daltons placed in series to the preservation apparatus was very efficient in eliminating the virus from perfusion solution in less than 2 hr. It can therefore be proposed that with the use of these molecular techniques, pulsatile perfusion coupled with additional viral depletion steps (dilution, and/or filtration) may allow the practical reduction of HCV transmission risk in recipient follow-up studies. The means are thereby presented for similar manipulation of other known or, as yet, unknown transmissible agents.
authors.affiliation: Department of Surgery, University of Miami School of Medicine, Florida.
authors.forename: K
authors.initials: K
authors.lastname: Zucker
authors.forename: R
authors.initials: R
authors.lastname: Cirocco
authors.forename: D
authors.initials: D
authors.lastname: Roth
authors.forename: L
authors.initials: L
authors.lastname: Olson
authors.forename: G W
authors.initials: GW
authors.lastname: Burke
authors.forename: J
authors.initials: J
authors.lastname: Nery
authors.forename: V
authors.initials: V
authors.lastname: Esquenazi
authors.forename: J
authors.initials: J
authors.lastname: Miller
mesh_terms: D001483:Base Sequence; D002102:Cadaver; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000382:microbiology*; D008107:Liver Diseases / Q000097:blood; D016031:Liver Transplantation / Q000009:adverse effects; D008969:Molecular Sequence Data; D009926:Organ Preservation / Q000379:methods*; D016133:Polymerase Chain Reaction; D011673:Pulsatile Flow; D012367:RNA, Viral / Q000032:analysis; D014462:Ultrafiltration; D014774:Virulence
chemical_list: D012367:RNA, Viral