PATIENTS receiving dialysis are at increased

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1 ORIGINAL INVESTIGATIONS Serologic and Virologic Profiles of Hepatitis C Infection in Renal Transplant Candidates Svetlozar N. Natov, MD, Johnson Y.N. Lau, MD, Beth A. Bouthot, MD, B.V.R. Murthy, MD, Robin Ruthazer, PhD, Christopher H. Schmid, PhD, Andrew S. Levey, MD, Brian J.G. Pereira, MD, DM, and the New England Organ Bank Hepatitis C Study Group The development of policies to prevent nosocomial transmission of hepatitis C virus (HCV) infection in hemodialysis units is critically dependent on the understanding of the relationship between tests for anti-hcv, HCV RNA, and HCV genotype and the patients clinical characteristics. We tested sera from all patients on the renal transplant waiting list at the New England Organ Bank between November 1986 and June 1990 for anti-hcv by a third-generation enzyme-linked immunosorbent assay (ELISA3) and a third-generation recombinant immunoblot assay (RIBA3). All ELISA3-positive sera were tested for HCV RNA by reverse transcriptase nested polymerase chain reaction, and the genotype was characterized by restriction fragment length polymorphism. Sera were available in 1,544 of 3,243 (48%) patients on the waiting list, of whom 287 (19%) tested positive for anti-hcv by ELISA3. Two hundred eighty-six randomly selected, anti-hcv negative patients served as controls. Compared with anti-hcv negative controls, anti-hcv positive patients had a longer duration since initiation of renal replacement therapy, higher number of previous kidney transplants and blood transfusions, higher proportion of patients with anti-hbc, history of liver disease, history of non-a, non-b hepatitis, and elevated serum alanine aminotransferase, and lower serum albumin concentrations. Of the 287 anti-hcv positive sera, 261 (91%) were reactive by RIBA3, 21 (7%) were indeterminate, and five (2%) were nonreactive. HCV RNA was detected in 224 of 275 (81%) ELISA3- positive patients, in whom additional sera were available. There were no significant differences in clinical or laboratory characteristics between ELISA3-positive patients with and without HCV RNA. Genotypes 1a, 1b, 2a, 2b, 3a, and 4 were present in 53%, 23%, 8%, 10%, 4%, and 2% of patients, respectively. Infection with one, two, or three different HCV genotypes was present in 92%, 7%, and 1%, respectively. There was no significant association between the type or number of HCV genotypes and RIBA3 reactivity. There were no major differences in clinical or laboratory characteristics between genotypes or between single and mixed infection. In summary, this study provides detailed information regarding the relationship between tests for anti-hcv, HCV RNA, and HCV genotypes and the clinical and laboratory characteristics of a large, well-characterized cohort of patients referred for renal transplant by the National Kidney Foundation, Inc. INDEX WORDS: Hepatitis C virus; viral infections; dialysis; dialysis infections; liver disease; hepatitis. From the Divisions of Nephrology and Clinical Care Research, New England Medical Center, Boston, MA, and Schering Plough Research Institute, Kenilworth, NJ. Received September 2, 1997; accepted in revised form December 19, Supported by the New England Organ Bank, Newton, MA; the Paul Teschan Research Fund of Dialysis Clinics Inc, Nashville, TN; a National Research Service Award (DK09450) from the National Institutes of Diabetes and Digestive and Kidney Diseases (to Dr Bouthot); the National Institutes of Health (DK48876); the Baxter Extramural Grant Program; and the Nephrology Clinical Research Fellowship of New England Medical Center and St Elizabeth s Hospital, Boston, MA. Address reprint requests to Brian J.G. Pereira, MD, DM, Division of Nephrology, New England Medical Center, Box 391, 750 Washington St, Boston, MA brian.pereira@es.nemc.org 1998 by the National Kidney Foundation, Inc /98/ $3.00/0 PATIENTS receiving dialysis are at increased risk of acquiring hepatitis C virus (HCV) infection from blood product transfusions or patient-to-patient transmission in hemodialysis units. 1 Consequently, the prevalence of HCV infection among patients receiving dialysis is consistently higher than that in the healthy population, and HCV is the single most important cause of chronic liver disease among dialysis patients. 1 Therefore, current attention has focussed on strategies to prevent patients from acquiring HCV infection while on dialysis, particularly by preventing the patient-to-patient transmission of HCV infection in hemodialysis units. The critical step in implementing policies to prevent the transmission of HCV infection in dialysis units involves the identification of patients with HCV infection. The HCV tests that are approved for clinical use detect antibodies to HCV (anti-hcv) rather than the HCV RNA itself. 2 The anti-hcv enzyme-linked immunosorbent assays (ELISAs) and recombinant immunoblot assays (RIBAs) detect nonneutralizing anti- 920 American Journal of Kidney Diseases, Vol 31, No 6 (June), 1998: pp

2 HCV IN RENAL TRANSPLANT CANDIDATES 921 bodies to recombinant HCV antigens. While ELISA detects antibodies to specific HCV antigens in a standard ELISA plate and is commonly used as a screening test, RIBA detects antibodies on a strip that is read visually, and it is considered a confirmatory test by virtue of its increased specificity. 2 The limited sensitivity of the firstand second-generation anti-hcv tests led to the development of the third-generation anti-hcv tests, which have incorporated multiple recombinant antigens from different parts of the HCV genome to overcome the limitations of the previous tests. Nonetheless, there is not a perfect concordance between anti-hcv and HCV RNA. Using reverse transcriptase polymerase chain reaction (PCR) to detect HCV RNA, several investigators have shown that a significant proportion of dialysis patients with a positive anti- HCV test can test negative for HCV RNA, and patients with a negative anti-hcv test can test positive for HCV RNA. 1 In addition, the characterization of a number of distinct HCV variants, designated as HCV types and subtypes, 3 with varying reactivity to anti-hcv tests has further confounded the interpretation of anti-hcv tests. Tests for anti-hcv, HCV RNA, and HCV genotypes require increasing levels of technical sophistication and resource allocation. Consequently, it is important to evaluate the relationship among the tests and the clinical characteristics of patients that test positive and negative for each test. We sought to study these relationships in a large cohort of patients referred for renal transplantation to 14 transplant centers served by the New England Organ Bank (NEOB) in the Northeastern United States. MATERIALS AND METHODS Patients The study population was drawn from all 14 transplant centers in the six New England states served by the NEOB. All patients on the renal transplant waiting list between November 1986 and January 1987 ( prevalent population) as well as those referred for renal transplantation between February 1987 and June 1990 ( incident population) were identified from the tissue typing laboratory records of the NEOB. Serum was available for anti-hcv testing in 394 of 655 (60%) prevalent patients and in 1,150 of 2,588 (44%) incident patients within the first 6 months of referral for transplantation. A control group of anti-hcv negative patients was randomly selected for the prevalent and incident cohorts separately. The anti-hcv negative controls were selected in a 1:1 ratio for each participating center,and those for whom information was not available were replaced with the next random number. Tests for HCV Infection Serum samples, stored at 70 C at the NEOB and the tissue typing laboratories at Brigham and Women s Hospital, Massachusetts General Hospital, and Maine Medical Center, were retrieved for HCV testing. Anti-HCV. The third-generation ELISA (ELISA3; Ortho HCV ELISA 3.0 Test System; Ortho Diagnostics System, Raritan, NJ) detects antibodies to recombinant HCV antigens (c22, c200, NS5) derived from the nonstructural (NS3, NS4, NS5) and core regions of the viral genome. The assay was performed according to the manufacturer s instructions. The third-generation RIBA (RIBA3; Chiron Corporation, Emeryville, CA) detects antibodies to two recombinant proteins (c33, NS5) and two synthetic peptide antigens (c22p, c100p), corresponding to the capsid, NS3, NS4, and NS5 regions of the viral genome. This assay was performed only on ELISA3-positive samples according to the manufacturer s instructions. HCV RNA. All ELISA3-positive serum samples were tested for HCV RNA by reverse transcriptase nested PCR with primers derived from the highly conserved 5 UTR region of the HCV genome. 4,5 HCV genotype. HCV genotypes were determined by restriction fragment length polymorphism of the 5 UTR PCR product. This method was chosen since amplification of the 5 UTR was shown to be the most efficient and, hence, genotyping is likely to be successful in the majority of the patients studied. Briefly, 5 UTR was reverse transcribed with outer primers (anti-sense: 5 -TCATGGTGCACGGTCTAC- GAGACCT-3 ; sense: 5 -CTGTAGGAACTACTGTCTT-3 ) and inner primers (anti-sense: 5 -CACTCGCAAGCACCTA- TCAGGCAGT-3 ; sense: 5 -TTCACGCAGAAAGCGTC- TAG-3 ) as described previously. 4,5 The amplification products were then digested by two restriction enzymes (HaeIII/ RsaI and Mval/Hinfl) to differentiate HCV into various major genotypes (types 1 to 6). Subtypes 1a/c and 1b were further differentiated by restriction with the enzyme BstUl. Subtypes 2a/c, 2b, 3a, and 3b were further differentiated by digestion with ScrFI. The nomenclature proposed by Simmonds et al 3 was used to classify HCV genotypes. In previous studies, we have shown that in samples obtained from patients in the United States, most of the 1a/c samples were 1a, and 2a/c samples were 2a (unpublished data). Hence, for the sake of simplicity, these samples were referred to as 1a and 2a, respectively. Clinical Information Clinical data were collected from the NEOB records, United Network for Organ Sharing Computer Services, databases of the tissue typing laboratories of Brigham and Women s Hospital, Massachusetts General Hospital, and Maine Medical Center, and hospital, transplant office, and dialysis unit medical records at the participating centers. Patients were enrolled into the study at the time of the first available serum between November 1986 and January 1987 (prevalent population) or within the first 6 months of referral

3 922 NATOV ET AL for transplantation (incident population). Clinical information collected included age at the time of enrollment into the study, gender, race, cause of end-stage renal disease (ESRD), presence of diabetes mellitus, year of initiation of dialysis, duration of renal replacement therapy, date of listing for renal transplantation at the NEOB, modes of dialysis, history of liver disease and non-a, non-b hepatitis (NANBH), number of blood transfusions, number of previous transplants, blood group, hepatitis B surface antigen, antibody to hepatitis B surface antigen (anti-hbs), antibody to hepatitis B core antigen (anti-hbc), and serum alanine aminotransferase (ALT) level and serum albumin at the time of enrollment into the study. Duration of renal replacement therapy was calculated from the date of initiation of dialysis (or previous transplantation, whichever was earlier) to the date of enrollment. Serum ALT was considered abnormal if it was greater than 2.5 times the upper limit of normal for the given laboratory. Statistical Analysis For discrete variables, the statistical significance of the difference between the groups was assessed by the Pearson s chi-square test or the Fisher s exact test when appropriate. For continuous variables, the Student s t-test was used to compare two groups and ANOVA was used to compare more than two groups. Pearson s correlation analysis was used for the calculation of correlation coefficients. Statistical significance was determined as a two-tail P value less than Calculations were performed using SAS/Stat (SAS Institute Inc, Cary, NC). RESULTS Prevalence of Anti-HCV and Clinical Characteristics of Prevalent and Incident Populations One hundred thirty-nine of 394 (35%) patients in the prevalent population tested positive for anti-hcv by ELISA3 compared with 148 of 1,150 (13%) in the incident population (P 0.001). Thus, in all, 287 of 1,544 (19%) patients were ELISA3 positive. Two hundred eighty-six randomly selected anti-hcv negative patients served as controls (122 incident and 164 prevalent patients). Thus, overall, 261 prevalent patients and 312 incident patients were included in the study (Table 1). Clinical Characteristics of Anti-HCV Positive and Negative Patients Referred for Renal Transplantation Compared with anti-hcv negative patients, anti-hcv positive patients had a longer duration since initiation of renal replacement therapy, higher number of previous kidney transplants and blood transfusions, higher proportion of patients with anti-hbc, history of liver disease, Table 1. Clinical Characteristics of the Prevalent and Incident Populations* Prevalent Population (n 261) Incident Population (n 312) P Value ESRD due to glomerular diseases 94/228 (41) 77/282 (27) Diabetes mellitus 29/229 (13) 69/286 (24) Duration of renal replacement therapy (mo) 70 (0-245) 14 (0-279) Mode of dialysis 0.02 HD 198/222 (89) 225/275 (82) PD 24/222 (11) 44/275 (16) HD and PD 0/222 (0) 6/275 (2) No. of previous transplants /224 (48) 205/283 (72) 1 87/224 (39) 61/283 (22) 2 25/224 (11) 12/283 (4) 3 4/224 (2) 5/283 (2) No. of blood transfusions /133 (3) 29/159 (18) /133 (20) 46/159 (29) /133 (17) 28/159 (18) 10 80/133 (60) 56/159 (35) Hepatitis B serology HBsAg 3/165 (2) 5/222 (2) 0.77 Anti-HBs 27/100 (27) 42/161 (26) 0.87 Anti-HBc 6/27 (22) 5/36 (14) 0.39 History of liver disease 52/209 (25) 47/275 (17) 0.04 History of NANBH 17/209 (8) 11/275 (4) 0.05 Elevated ALT 7/127 (6) 11/161 (7) 0.65 Albumin (g/dl) 3.8 ( ) 3.9 ( ) 0.27 Abbreviations: HD, hemodialysis; PD, peritoneal dialysis. *Not all patients had all data available. Therefore, the total number of patients for whom data for each variable are available is less than the number of patients in each group. Data for continuous variables are presented as median (range) and for discrete variables as fraction (%). Patients on the renal transplant waiting list between November 1986 and January Patients referred for renal transplantation between February 1987 and June Pearson s chi-square test or Fisher s exact tests for discrete variables and Student s t-test for continuous data. history of NANBH and elevated serum ALT, and lower serum albumin levels (Table 2). Relationship Among Third-Generation Screening and Confirmatory Anti-HCV Tests Of the 287 anti-hcv positive sera, 261 (91%) were reactive by RIBA3, 21 (7%) were indeterminate, and five (2%) were nonreactive.

4 HCV IN RENAL TRANSPLANT CANDIDATES 923 Table 2. Clinical Characteristics of Anti-HCV Positive and Negative Patients Referred for Renal Transplantation* Anti-HCV Positive (n 287) Anti-HCV Negative (n 286) P Value ESRD due to glomerular diseases 81/226 (36) 89/283 (31) 0.30 Duration of renal replacement therapy (mo) 68 (0-283) 14 (0-214) Mode of dialysis 0.66 HD 196/226 (87) 228/271 (84) PD 28/226 (12) 39/271 (14) HD and PD 2/226 (1) 4/271 (2) No. of previous transplants /226 (48) 204/270 (76) 1 83/226 (36) 55/270 (20) 2 29/226 (13) 8/270 (3) 3 6/226 (3) 3/270 (1) No. of transfusions /166 (5) 24/127 (19) /166 (15) 49/127 (39) /166 (19) 18/127 (14) /166 (61) 36/127 (28) Hepatitis B serology HBsAg 4/190 (2) 4/196 (2) 0.96 Anti-HBs 41/131 (31) 28/130 (22) 0.07 Anti-HBc 6/18 (33) 5/45 (11) 0.04 History of liver disease 86/215 (40) 13/276 (5) History of NANBH 18/287 (6) 8/286 (3) 0.05 Elevated ALT 14/105 (13) 4/183 (2) Albumin (g/dl) 3.8 ( ) 4.0 ( ) Abbreviations: HD, hemodialysis; PD, peritoneal dialysis. *Not all patients had all data available. Therefore, the total number of patients for whom data for each variable are available is less than the number of patients in each group. Data for continuous variables are presented as median (range) and for discrete variables as fraction (%). Pearson s chi-square test for discrete variables and Student s t-test for continuous data. Anti-HCV Positive Patients With and Without HCV RNA by PCR Two hundred seventy-five ELISA3-positive patients (138 from the prevalent and 137 from the incident population) had serum available for PCR testing. HCV RNA was detected in 112 (81%) of 138 prevalent patients, 112 (82%) of 137 incident patients, and 224 (81%) of 275 in the total group of patients. Among anti-hcv positive patients, there were no significant differences between PCR-positive and -negative patients with respect to the demographic, clinical, and laboratory characteristics shown in Table 1. Among ELISA3-positive patients, RIBA3 was reactive, indeterminate, or nonreactive in 93%, 7%, and less than 1%, respectively, among those with HCV RNA compared with 86%, 8%, and 6%, respectively, among those without HCV RNA (P 0.01). Distribution of Different HCV Genotypes Genotyping was performed in all 224 HCV RNA-positive patients. The distribution of different HCV genotypes among the prevalent and incident populations is shown in Table 3. Genotype 1a was the most frequent HCV genotype in both groups, followed by genotype 1b. Genotype 4 was found exclusively among the incident population. Among patients infected with a single genotype, there were no significant differences in the distribution of the different HCV genotypes between the prevalent and incident groups (P 0.11). Infection with a single genotype was the most common pattern in both the prevalent and incident populations. Interestingly, infection with two or more genotypes was slightly but significantly more common in the incident than the prevalent population (P 0.03). No specific HCV genotype pattern could be associated with mixed (double or triple) infection. There was no statistically significant difference between the Table 3. Distribution of Different HCV Genotypes Among Patients Referred for Renal Transplantation Genotype Prevalent Population*: Patients 112; Genotypes 116 (%) Incident Population : Patients 112; Genotypes 128 (%) Overall: Patients 224; Genotypes 244 (%) Genotype 1a 61 (53) 68 (53) 129 (53) 1b 34 (29) 22 (17) 56 (23) 2a 6 (5) 13 (10) 19 (8) 2b 12 (10) 14 (11) 26 (10) 3a 3 (3) 6 (5) 9 (4) 4 0 (0) 5 (4) 5 (2) Genotypes per patient One 108 (96) 98 (87) 206 (92) Two 4 (4) 12 (11) 16 (7) Three 0 (0) 2 (2) 2 (1) *Patients on the renal transplant waiting list between November 1986 and January Patients referred for renal transplantation between February 1987 and June 1990.

5 924 NATOV ET AL various HCV genotypes or between patients with single, double, or triple infections with regard to RIBA3 reactivity. Table 4 shows the distribution of the HCV genotypes based on the year of initiation of renal replacement therapy in 181 PCR-positive patients for whom this information was available. There was no significant change in the proportion of the two most common HCV genotypes (1a and 1b) among patients who initiated renal replacement therapy during different time periods. Clinical Characteristics of Patients With Respect to the Type and Number of Infecting HCV Genotypes Among patients with single-genotype infection, we related infection with 1a, 1b, or others (non 1a,1b) to demographic, clinical, and laboratory characteristics. No statistically significant differences were detected among those groups except for race and presence of HBsAb. Among those with 1a, 1b, and non 1a,1b, nonwhite patients constituted 30%, 49%, and 19%, respectively (P 0.02) and HBsAb was present in 36%, 38%, and 0%, respectively (P 0.02). Patients with mixed infections had significantly lower mean serum albumin compared with those with single-genotype infection ( v ; P 0.04). There were no other significant differences between the patients with single- or mixed-genotype infections. DISCUSSION Using PCR as the gold standard for active HCV infection, the third-generation anti-hcv tests have been shown to have an improved Table 4. Distribution of Different HCV Genotypes Among Patients Referred for Renal Transplantation by Year of Initiation of Renal Replacement Therapy Year of Initiation of Renal Replacement Therapy (%) 1975 (n 47) (n 44) (n 63) (n 47) 1a 27 (58) 25 (58) 31 (50) 27 (58) 1b 9 (19) 9 (20) 17 (27) 8 (17) 2a 3 (6) 3 (7) 2 (3) 5 (11) 2b 6 (13) 5 (11) 8 (12) 4 (8) 3a 2 (4) 1 (2) 3 (5) 2 (4) 4 0 (0) 1 (2) 2 (3) 1 (2) sensitivity and specificity compared with the first- and second-generation tests. 6,7 However, limited data are available on the performance of these tests in patients with ESRD and the clinical characteristics of patients who test positive. Using ELISA3, we found a 19% prevalence of anti-hcv among dialysis patients referred for renal transplantation to the 14 transplant centers served by the NEOB between 1986 and Our observation is in accordance with the reported prevalence of anti-hcv in other dialysis centers in the United States. Indeed, among the 27,086 patients from dialysis centers participating in the National Surveillance of Dialysis- Associated Diseases in the United States conducted by the Centers for Disease Control and Prevention, the prevalence of anti-hcv by ELISA2 was 8.1%, with a range of 0% to 51% among centers with 40 patients. 8 The prevalence of anti-hcv in the prevalent population (35%) was significantly higher than in the incident population (13%). Indeed, the prevalent patients had been receiving dialysis longer and had received higher numbers of blood transfusions and previous transplants, all risk factors for acquiring HCV infection. The lower prevalence of anti-hcv among patients referred for renal transplantation between 1987 and 1990 is also likely to reflect the declining trend in the incidence and prevalence of NANBH that was observed even before the discovery of HCV. 8 The incidence of NANBH among dialysis patients in the United States has declined from 1.7% in 1982 to 0.5% in This decline was probably the result of exclusion of blood products that tested positive for surrogate markers of NANBH, such as elevated ALT and anti-hbc, and the implementation of strategies to reduce transmission of blood-borne infections in dialysis units. 9,10 The discovery of HCV in 1989, the advent of testing of blood products for anti-hcv, and the implementation of more rigorous infection-control measures to prevent nosocomial transmission within dialysis units have led to a further decline in the incidence and prevalence of HCV infection. Indeed, the incidence of NANBH among dialysis patients in the United States declined further, from 0.5% in 1990 to 0.4% in Likewise, among member nations in the European Dialysis and Transplant Association, the prevalence of anti-hcv declined from

6 HCV IN RENAL TRANSPLANT CANDIDATES % in 1992 to 17.7% in 1993, 11,12 and among hemodialysis units in Portugal, the incidence of HCV infection declined from 11.2% in 1991 to 7.2% in 1992 and 6.5% in Anti-HCV positive ESRD patients in this study had a longer duration of renal replacement therapy, higher number of previous transplants and blood transfusions, higher proportion of patients with anti-hbc, history of liver disease, history of NANBH, elevated serum ALT, and lower serum albumin levels. The association between anti-hcv and a longer duration of renal replacement therapy and higher number of blood transfusions observed in this study has been previously reported. 14 The significant association between anti-hcv and the number of previous transplants could be attributed to the increased risk of acquiring HCV infection from the organ donor and/or perioperative blood transfusions in the era before the discovery of HCV. Indeed, we have previously shown that HCV is transmitted by organ transplantation. 15 Interestingly, serum albumin levels were significantly lower among anti-hcv positive patients compared with anti- HCV negative patients. The lower serum albumin levels among anti-hcv positive patients could be due to decreased liver synthetic function associated with chronic hepatitis C. Alternatively, it may reflect malnutrition among patients who have been on dialysis longer and consequently were at increased risk of acquiring HCV infection. Finally, hypoalbuminemia may be a surrogate marker for immune dysfunction and inability to clear HCV among ESRD patients exposed to the virus. HCV RNA was detected in 81% of the ELISA3-positive patients, confirming previous observations that the presence of anti-hcv does not necessarily imply the presence of HCV RNA in the serum. We did not observe significant differences in demographic, clinical, or laboratory characteristics between anti-hcv positive patients with and without HCV RNA by PCR. Although HCV RNA-positive patients had a higher rate of RIBA3 reactivity compared with HCV RNA-negative patients, RIBA3 did not accurately predict the absence of HCV RNA. Presence of anti-hcv in the absence of HCV RNA probably results from persistence of antibody to HCV even after the virus has been cleared, passively acquired anti-hcv from blood transfusions, 16 levels of HCV RNA below the limit of detection, 17 sequestration of the virus at sites other than the blood stream, 14,18 intermittent viremia, 19 or false-positive results due to nonspecific reactions. Hence, it remains currently unclear whether anti-hcv positive patients who are PCR negative are indeed free from infection. This uncertainty has confounded policies concerning patient isolation in dialysis units. Several reports have emphasized genotyperelated differences in disease severity, clinical outcomes, and response to interferon among nonrenal patients with chronic hepatitis C. 20,21 In patients without renal failure, some investigators have observed that infection with genotype 1b was associated with more severe liver disease (cirrhosis and hepatocellular carcinoma), a greater cytopathic effect in liver transplant recipients, and a lower rate of response to interferon treatment. 21,22 In contrast, others have observed that genotype 2 was associated with more serious liver disease (chronic active hepatitis or cirrhosis), despite significantly lower levels of circulating HCV RNA compared with other genotypes. 23 In addition, a long-term sustained response to interferon- treatment occurred less frequently in patients infected with genotype 1 (7%) than in patients infected with other genotypes (40%). 23 We observed that genotypes 1a, 1b, 2a, 2b, 3a, and 4 were present in 53%, 23%, 8%, 10%, 4%, and 2% of patients, respectively. The genotype distribution among our patients reflects the distribution among nonrenal patients in the United States, where genotype 1a and 1b are the most commonly encountered genotypes. 23 Interestingly, the distribution of HCV genotypes has remained relatively constant among patients who started renal replacement therapy during the following periods: before 1975, 1975 to 1980, 1981 to 1985, and 1986 to This observation is in contrast to the results from France, where genotypes 1a and 2a appeared in the 1970s, while genotypes 3a, 4a, and 5a emerged in the 1980s. 24 The French investigators also noted that the prevalence of genotype 1b, which was the cause for more than two thirds of HCV infection among the patients undergoing dialysis before 1977, decreased during the study period, while the prevalence of genotype 2a increased and became the most common current cause of HCV infection in their dialysis population. In contrast to

7 926 NATOV ET AL these observations, Mahaney et al 23 reported that in nonrenal patients with chronic hepatitis C in the United States, genotype 1 was the most frequent HCV genotype (74% of cases) at any time. These investigators failed to demonstrate any substantial change in the spectrum of the infecting HCV genotypes over a period of 2 to 20 years and, like us, were unable to show differences among patients infected with different HCV genotypes. 23 Studies in humans and animals with HCV infection have revealed a lack of protective immunity against reinfection (new infection after the previous infection has cleared) with the same or different genotype 25,26 or superinfection (infection with a new genotype in the presence of a preexisting infection). 27 Reinfection has been reported in thalassemic children after multiple blood transfusions. 26 These reports indicate that HCV infection does not induce protective immunity, and that both reinfection and superinfection can occur. The newly introduced HCV genotype may either replace ( take over ), be eliminated, or coexist with the predecessor HCV genotype. The clinical implications of each of these virologic outcomes are currently unclear. In our study, infection with one, two, or three different HCV genotypes was present in 92%, 7%, and 1% of patients, respectively. There were no significant differences in demographic, clinical, or laboratory characteristics between patients infected with single or mixed infections, with the exception of a lower serum albumin among patients with mixed infection. The lower serum albumin levels among patients with mixed infections could be due to a greater degree of liver dysfunction or to malnutrition among patients who have been receiving dialysis longer and are consequently at increased risk of acquiring multiple HCV genotype infection. In summary, this study provides detailed information regarding the relationship between tests for anti-hcv, HCV RNA, and HCV genotypes and the demographic, clinical, and laboratory characteristics of a large, well-characterized cohort of patients referred for renal transplantation. REFERENCES 1. Pereira BJG, Levey AS: Hepatitis C virus infection in dialysis and renal transplantation. Kidney Int 51: , Natov SN, Pereira BJG: Hepatitis C infection in patients on dialysis. Semin Dial 7: , Simmonds P, Alberti A, Alter HJ, Bonino F, Bradley DW, Brechot C, Brouwer JT, Chan S-W, Chayama K, Chen D-S, Choo Q-L, Colombo M, Cuypers HTM, Date T, Dusheiko GM, Esteban JI, Fay O, Hadziyannis SJ, Han J, Hatzakis A, et al: A proposed system for the nomenclature of hepatitis C virus genotypes. Hepatology 19: , Lau JYN, Davis GL, Orito E, Qian KP, Mizokami M: Significance of antibody to the host cellular gene derived epitope GOR in chronic hepatitis C virus infection. J Hepatol 17: , Lau JYN, Davis GL, Kniffen J, Qian KP, Urdea MS, Chan CS, Mizokami M, Neuwald PD, Wilber JC: Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 341: , Couroucé AM, Bouchardeau F, Chauveau P, Le Marrec N, Girault A, Zins B, Naret C, Poignet JL: Hepatitis C virus (HCV) infection in haemodialysed patients: HCV-RNA and anti-hcv antibodies (third-generation assays). Nephrol Dial Transplant 10: , Soffredini R, Rumi M, Lampertico P, Aroldi A, Tarantino A, Ponticelli C, Colombo M: Increased detection of antibody to hepatitis C virus in renal transplant patients by third-generation assays. Am J Kidney Dis 28: , Tokars J, Alter MJ, Favero MS, Moyer LA, Miller E, Bland L: National surveillance of hemodialysis associated diseases in the United States, ASAIO J 40: , Alter MJ, Favero MS, Maynard JE: Impact of infection control strategies on the incidence of dialysis-associated hepatitis in the United States. J Infect Dis 153: , Alter MJ, Favero MS, Moyer LA, Bland LA: National surveillance of dialysis-associated diseases in the United States, ASAIO Trans 37:97-109, Geerlings W, Tufveson G, Ehrich JHH, Jones EH, Landais P, Loriat C, Mallick NP, Margreiter R, Raine EE, Salmela K, Selwood NH, Valderrabano F: Report on the management of renal failure in Europe, XXIII. Nephrol Dial Transplant 9:6-25, Valderrabano F, Jones EHP, Mallick NP: Report on management of renal failure in Europe, XXIV, Nephrol Dial Transplant 10:1-25, 1995 (suppl 5) 13. Pinto dos Santos J, Loureiro A, Cendoroglo M, Pereira BJG: Impact of dialysis room and reuse strategies on the incidence of HCV infection in HD units. Nephrol Dial Transplant 11: , Dussol B, Chicheportiche C, Cantaloube JF, Roubicek C, Biagini P, Berthézenè P, Berland Y: Detection of hepatitis C infection by polymerase chain reaction among hemodialysis patients. Am J Kidney Dis 22: , Pereira BJG, Milford EL, Kirkman RL, Levey AS: Transmission of hepatitis C virus by organ transplantation. N Engl J Med 325: , Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo Q-L: Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-a, non-b hepatitis. N Engl J Med 321: , DuBois DB, Gretch D, dela Rosa C, Lee W, Fine J,

8 HCV IN RENAL TRANSPLANT CANDIDATES 927 Blagg CR, Corey L: Quantitation of hepatitis C viral RNA in sera of hemodialysis patients: Gender-related differences in viral load. Am J Kidney Dis 24: , Willems M, Peerlinck K, Moshage H, Deleu I, Van den Eyden C, Vermylen J, Yap SH: Hepatitis C virus-rnas in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C: Evidence for viral replication in peripheral blood mononuclear cells. J Med Virol 42: , Farci P, Alter HJ, Wong D, Miller RH, Shih JW, Jett B, Purcell RH: A long-term study of hepatitis C virus replication in non-a, non-b hepatitis. N Engl J Med 325:98-104, McOmish F, Chan SW, Dow BC, Gillon J, Frame WD, Crawford RJ, Yap PL, Follett EA, Simmonds P: Detection of three types of hepatitis C virus in blood donors: Investigation of type-specific differences in serologic reactivity and rate of alanine aminotransferase abnormalities. Transfusion 33:7-13, Yoshioka K, Kakumu S, Wakita T, Ischikawa T, Itoh Y, Takayangi M, Higashi Y, Shibata M, Morishima T: Detection of hepatitis C virus by polymerase chain reaction and response to interferon-alpha therapy: Relationship to genotypes of hepatitis C virus. Hepatology 16: , Feray C, Grigou M, Samuel D, Okamoto H, Reynes M, Mishiro S, Gigou M, Bismoth H, Brechot C: HCV type II has a more pathogenic course after liver transplantation. Hepatology 18:59, 1993 (abstr) 23. Mahaney K, Tedeschi V, Maertens G, Di Bisceglie AM, Vergalla J, Hoofnagle JH, Sallie R: Genotypic analysis of hepatitis C virus in American patients. Hepatology 20: , Pol S, Thiers V, Nousbaum JB, Legendre C, Berthelot P, Kreis H, Brechot C: The changing relative prevalence of hepatitis C virus genotypes: Evidence in hemodialyzed patients and kidney recipients. Gastroenterology 108: , Farci P, Alter HJ, Govindarajan S, Wong DC, Engle R, Lesniewski RR, Mushahwar IK, Desai SM, Miller RH, Ogata N, Purcell RH: Lack of protective immunity against reinfection with hepatitis C virus. Science 258: , Lai ME, Mazzoleni AP, Argiolu F, De Virgilis S, Balestrieri A, Purvell RH, Cao A, Farci P: Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children. Lancet 343: , Kao JH, Chen PJ, Lai MY, Chen DS: Superinfection of heterologous hepatitis C virus in a patient with chronic type C hepatitis. Gastroenterology 105: , 1993