Letter to the Editor Nephron 1994:68:140 Detection of Hepatitis C Virus in Dialysate and in Blood Ultrafiltrate of HCV-Positive Patients M. Sampietroa G. Grazianib S. Bcidalamentib S. Salvador/a R. CaldarellR G. Comob G. Fiorellia Istituto di Medicina Interna e Fisiopatologia Medica, Universitá di Milano e Divisione di Nefrologia e Dialisi, IRCCS Ospedale Maggiore Policlinico, Milano, Italia Dear Sir, Hepatitis C virus (HCV) is a major cause of hepatitis in haemodialysis patients. The prevalence of anti-HCV antibodies increases significantly with increasing age and fre quency of haemodialysis. The analysis of known risk factors such as blood transfu sions, status of hepatitis Band HIV infection, and intravenous drug addiction appears to leave an unaccounted residual risk which is directly related to the duration of exposure to dialytic treatment [1-3]. Therefore other, as yet undefined, parenteral routes of trans mission of HCV may exist in the haemodial ysis setting. With the aim of investigating unrecog nized possible routes of HCV transmission in dialysis patients we examined samples of fluid collected at the dialysate outlet of filtres and samples of blood ultrafiltrate during the dialytic treatment of HCV-positive patients with the polymerase chain reaction (PCR) for the presence of HCV RNA. Four 100-ml samples were collected at the end of sessions (three dialysates from poly- methylmetacrylate, cuprophane and polysul- phone membranes, respectively, and one ultrafiltrate from polysulphone). All samples were immediately processes by centrifugation through Centriprep 100 (Amicon Division, Beverly. Mass., USA) cartridges to obtain a 100-fold concentration. RNA extraction was performed with the acid guanidinium thiocy- anate-phenol-chloroform method [4] with the addition of I ug glycogen as a carrier prior to isopropanol precipitation. Complementary DNA (cDNA) synthesis (Promega Corpora tion, Madison, Wise., USA) was primed from the core region of HCV RNA. Nested PCR was performed using primer sequences lo cated in 5' untranslated region (235 bp prod uct, nt 275-40) [5] and in the core region (143 bp product, nt 148-291) [6] of the viral genome. Strict precautions to prevent contamination were adopted: negative controls were intro duced during RNA extraction and processed throughout cDNA synthesis and PCR. In two samples, one dialysate and one ultrafiltrate, both from polysulphone mem branes, we obtained PCR products of the appropriate size visualized on ethidium bro mide-stained polyacrylamide gels. In both cases we found positivity for the 5' untrans lated region as well as for a core HCV type 11 sequence, consistent with the viral subtype previously identified in the patients. The passage of viral particles across dialy sis membranes is likely to occur through microruptures of capillaries giving rise to leakages of blood, below the sensitivity of the blood leak detector, into the dialysis fluid. They are likely to happen as the result of intradialytic mechanical stress of membranes and/or microclotting of some hollow fibres followed by their obstruction in a system with high blood flow and pressure. This event could be influenced by many factors, such as the type and preparation of membrane, type and duration of dialysis, transmembrane pressures, incorrect use of heparin and titre of viraemia in the dialysis patient, all of which will require further evaluation. Our findings indicate the existence of a potential risk of cross-infection of HCV by dialysis fluids in patients utilizing high flux membranes with backfiltration of dialysis fluid into the blood. This would suggest the utility to adopt precautions such as the segre gation of machines used for HCV-positive patients and disinfection of machines after each dialytic session. References 1 Machida J, Yamaguchi K, Ueda S, Yoshida M, Kusamoio Y, Nishimura Y. Futami G, Ishii T. Watanabe T, Takatsuki K: High incidence of hepatitis C virus antibodies in hemodialysis pa tients. Nephron 1992:60:117-118. 2 Jonas M. Zilleruelo GE. LaRue S, Abitbol C, Strauss J, Lu Y: Hepatitis C infection in a pedi atric dialysis population. Pediatrics 1992:89: 707-709.’ 3 Knudsen F. Wantzin P. Rasmussen K, La- defoged SD, Lokkegard N, Rasmussen LS, Lassen A, Krogsgaard K: Hepatitis C in dialysis patients: Relationship to blood transfusions, di alysis and liver disease. Kidney Int 1993:43: 1353-1356. 4 Chomczynski P. Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocya nate-phenol-chloroform extraction. Anal Bio- chem 1987;162:156-169. 5 Bukh J, Purcell RH. Miller RH: Sequence anal ysis of the 5' noncoding region of hepatitis C virus. Proc Natl Acad Sci USA 1992:89:4942 4949. 6 Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y. Sugai Y, Tanaka T, Sato K, Tsuda F, Miyakawa Y, Mayumi M: Typing hepatitis C virus by polymerase chain reaction with type- specific primers: Application to clinical surveys and tracing infectious sources. J Gen Virol 1992; 73:673-679. Dr. Maurizio Sampietro © I994S. Karger AG, Basel I RCCS Ospcdalc Maggiore Policlihico, Padiglione LITTA 0028-2766/94 via F. Sforza, 35 0681 OI40S8.00/0 I -20122 Milano (Italy)