They were all recruited from a single dialysis unit in Pavia,. Italy. .... patients were hepatitis B virus surface antigen (HBsAg) positive. ..... Braun, W. E. 1990.
Vol. 31, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1993, p. 2913-2917
0095-1137/93/112913-05$02.00/0 Copyright ©) 1993, American Society for Microbiology
Virological Features of Hepatitis C Virus Infection in Hemodialysis Patients ENRICO SILINI,1 FULVIA BONO,' ANTONELLA CERINO,2 VALTER PIAZZA,3 ENRICO AND MARIO U. MONDELLI2* Dipartimento di Patologia Umanal and Istituto di Clinica delle Malattie Infettive, 2 IRCCS Policlinico S. Matteo and University of Pavia, and Divisione di Nefrologia e Dialisi, Fondazione Clinica del Lavoro,3 Pavia, Italy
SOLCIA,1
Received 4 May 1993/Returned for modification 1 July 1993/Accepted 3 August 1993
The clinical and epidemiological relevance of circulating antibodies to hepatitis C virus (HCV) in
hemodialysis patients is uncertain, since clinical signs of infection are often mild or absent, with alanine aminotransferase (ALT) values that are virtually always normal, and liver biopsies are only rarely performed.
Determination of HCV RNA in serum is therefore critical for distinguishing chronic HCV infection from previous exposure to the virus. We studied HCV viremia by reverse transcription polymerase chain reaction (RT-PCR) in the 5'-noncoding region of the viral genome in 77 dialysis patients who were screened for anti-HCV by a second-generation enzyme-linked immunosorbent assay (the enzyme immunoassay II; Ortho HCV, 2nd generation, Ortho Diagnostic Systems Raritan, N.J.) and a second-generation recombinant immunoblot assay (Chiron Corporation and Ortho Diagnostic Systems) and prospectively evaluated for ALT elevations over a period of 5 years. Of 77 patients tested, 29 (38%) had active infection as shown by a positive PCR assay result, and of these, 26 were anti-HCV positive. Although a good correlation was found between circulating anti-HCV and HCV RNA in serum, 10 (28%) of 36 anti-HCV-positive patients were HCV RNA negative by PCR, suggesting either low levels of viremia or past exposure to HCV and subsequent recovery. On the other hand, 3 (7.3%) of 41 anti-HCV-negative patients had HCV RNA in their sera, indicating seronegative HCV infection. The ALT level had no predictive value for HCV infection, because it was repeatedly normal in 18 (62%) of 29 viremic patients. HCV genotyping was also performed and indicated that all four known genotypes of HCV were present in our group. In conclusion, serological assays are reliable for detecting exposure to HCV in hemodialysis patients; however, direct identification of the viral genome is required to document current infection. a larger cohort of 277 patients (16) screened for anti-HCV by a second-generation enzyme-linked immunosorbent assay (ELISA) (the enzyme immunoassay II [EIA II]) and a second-generation recombinant immunoblot assay (RIBA II)
Hemodialysis patients carry a variable risk of developing hepatitis C virus (HCV) infection, and a major proportion of them has been exposed to the virus, as documented by several serological surveys (9, 13, 15-17, 28). Anti-HCV seroprevalences in the different reported series range from 12 to 29% to 22 to 55%, depending on the use of first- or second-generation immunoassays, respectively, and they are independent of the prevalences of anti-HCV in the general population from the same geographic area (17). Duration of hemodialysis, past history of blood transfusions, and alanine aminotransferase (ALT) elevations correlate with higher rates of anti-HCV positivity (12, 15-17, 28). However, the clinical and epidemiological relevance of the presence of anti-HCV in hemodialysis patients is not clear. Clinical signs of acute infection are often mild or absent, and there is a substantial risk of developing chronic liver disease which is usually asymptomatic for several years (19). In addition, only limited information is available on the underlying liver pathology, since liver biopsies are only rarely performed in these patients. The ability to detect circulating HCV RNA in serum by reverse transcription polymerase chain reaction (RT-PCR) (10, 27) provides a mean of identifying patients with active infection because of the high sensitivity of the technique, allowing verification of the efficacy of serological assays in screening exposed patients. We applied RT-PCR to a study of HCV viremia in 77 dialyzed patients selected from *
and prospectively evaluated the patients for ALT elevations over a period of 5 years. To test the value of serological and
biochemical indices predictive of active infection, both antiHCV-positive and -negative patients, with or without raised liver enzyme levels, were studied. Sera from 26 viremic patients were also investigated for the prevalence of HCV genotypes I to IV (4) by using a type-specific primer system based on sequences of the core gene (17). MATERIALS AND METHODS
Patients. Seventy-seven patients (43 males and 34 females; median age, 62 years; age range, 16 to 80 years) who were treated with hemodialysis for terminal renal failure for a median of 76 months (range, 9 to 288 months) were studied. They were all recruited from a single dialysis unit in Pavia, Italy. ALT levels were checked bimonthly and were retrospectively analyzed for 5 years (1988 to 1992). At least one ALT elevation of more than twice the upper limit of normal was found in 24 (31%) patients, 16 (21%) of whom showed multiple peaks. However, at the time of the study, all but one patient had normal ALT levels. ALT elevations were always moderate and never exceeded four times the upper limit of normal. Anti-HCV detection. All patients were screened by an EIA
Corresponding author. 2913
2914
SILINI ET AL.
II that detects antibodies to core antigen and a fusion of the c100-3 and c33-c antigens (c200) of HCV (Ortho HCV, 2nd generation; Ortho Diagnostic Systems, Raritan, N.J.) according to the manufacturer's instruction. Tests were performed in duplicate on aliquots stored frozen at -20°C and used only once. For each patient, at least 10 serological tests were done during the study period. For most patients, data were also available for anti-clOO-3 antibodies detected by a first-generation anti-HCV immunoassay (EIA I) (Ortho Diagnostic Systems). Anti-HCV immunoreactivity by EIA II was confirmed for all patients by RIBA II (Chiron Corporation and Ortho Diagnostic Systems), which uses four recombinant HCV antigens (5-1-1, clOO-3, c33c, and c22-3) fused to human superoxide dismutase. HCV RNA detection. Newly collected serum samples stored frozen at -70°C were used for HCV RNA extraction, which was performed by the acid guanidinium thiocyanatephenol-chloroform method (8), following concentration of serum by polyethylene glycol precipitation (10). Primers used for retrotranscription and nested PCR amplification were localized in conserved sequences of the 5'-noncoding region of the viral genome (3, 11) and were synthesized on an oligosynthesizer (model 391; Applied Biosystem Inc., Foster City, Calif.). Primers sequences were as follows: sense external primer, 5'-CGGAACCGGTGAGTACACCGG-3'; antisense external primer, 5'-CACTCGCAAGCACCCTAT CAGG-3'; sense internal primer, 5'-ACGACCGGGTCCTT TCTTGG-3'; and antisense internal primer, 5'-CGCGAC CCAACACTACTCGG-3'. RNA from 80 ,ul of serum was reverse transcribed for 1 h at 37°C in a 20-pu reaction volume containing 50 mM KCl, 20 mM Tris-HCl (pH 8.4), 2.5 mM MgCl2, 1 mM (each) deoxynucleoside triphosphates, 20 U of RNAsin (Promega, Madison, Wis.), 10 pM antisense primer, and 200 U of mouse mammary leukemia virus reverse transcriptase (GIBCO-Bethesda Research Laboratories, Gaithersburg, Md.). Ten microliters of the cDNA mixture was amplified with the external primer pair in a reaction volume of 50 ,ul containing 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 0.2 mM (each) deoxynucleoside triphosphates, 50 pM each primer, and 0.5 U of Taq polymerase (Perkin-Elmer, Norwalk, Conn.). Cycling parameters were 94°C for 1 min, 55°C for 3 min, and 72°C for 3 min for 5 cycles and 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min for 35 cycles. First-round PCR products were analyzed by gel electrophoresis on a 10% polyacrylamide gel in Tris borate buffer stained with ethidium bromide. Negative samples and serial dilutions of a reference positive control were subjected to a second round of amplification with the inner primers pair at 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min for 5 cycles and 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 20 cycles. The sensitivity of the method, as determined in reconstruction experiments with HCV cloned sequences, was 1 to 10 copies of template. To avoid contamination by PCR product carryover and false-positive results, samples were prepared in a separate laboratory from that in which the reactions were done; we also used reagents that were divided into aliquots, aerosol-free tips, and all other recommended precautions (14). HCV genotyping. HCV genotyping was performed by the method described by Okamoto et al. (18), which uses amplification by PCR of core gene sequences with universal and four type-specific primers to generate DNA fragments with a size specific for each of the four HCV types. Type IIIspecific primer was modified (5'-GCCCCATGAAGGGC GAGAAC-3') because of small nucleotide differences of type III Italian isolates compared with those from Japan (la).
J. CLIN. MICROBIOL.
TABLE 1. EIA II, RIBA II, and HCV RNA status in 77 hemodialysis patients Test and result (no. of patients)
EIA II positive (36) EIA II negative (41)
Total (77)
No. of patients
RIBA II positive
RIBA II indeterminate
HCV RNA positive
29
7
26 3
29
7
29
Statistical analysis. The Wilcoxon two-sample test, the chi-square test, or Fisher's exact test was used as appropriate.
RESULTS Anti-HCV detection. A total of 36 (46%) patients were found to be positive for anti-HCV by a second-generation ELISA (EIA II). For 33 of them, optical density values were greater than 3, while lower values were found in only 3 patients. One patient showed decreasing optical density values during follow-up. Twenty-nine patients were also positive by a second-generation immunoblot assay (RIBA II) and 7 were indeterminate (Table 1; for detailed immunoreactivities, see Table 4). Five of seven RIBA II indeterminate patients were hepatitis B virus surface antigen (HBsAg) positive. Patients positive for anti-HCV had a longer history of dialysis (median dialysis time, 108 versus 58 months; P < 0.005) and higher rates of ALT elevations (44 versus 19%; P < 0.04) than those who were anti-HCV negative. Sixteen patients were HBsAg positive. HCV RNA detection. HCV RNA was detected by RT-PCR in the serum of 29 of 77 (37.6%) patients; 26 (89.6%) of them were also positive for anti-HCV by EIA II. The remaining 3 (10.3%) were anti-HCV negative. Prevalences of viremic patients in anti-HCV-positive versus anti-HCV-negative groups were 72.2 and 7.3%, respectively. Positivity for HCV RNA was markedly different in anti-HCV-positive patients according to their HBsAg status. Thus, 23 (85%) of 27 HBsAg-negative patients were viremic, whereas 3 (33%) of 9 HBsAg-positive patients were viremic (P < 0.007). All three anti-HCV-negative patients with viremia gave a positive test result only after the second amplification step, suggesting a low viral titer, and conversely, all 26 anti-HCV-positive patients were positive after the first amplification. Nine of the 10 anti-HCV-positive, HCV RNA-negative patients had A492 values of greater than 3. The remaining patients had an A492 value of 2.503 and the result was indeterminate by RIBA II. Comparison of ALT levels, anti-HCV testing, and HCV RNA. Comparisons of ALT elevations, HCV antibody testing by EIA II, and HCV RNA status are shown in Table 2. As expected, a good correlation was found between a positive anti-HCV test result and the presence of HCV RNA by RT-PCR. However, ALT levels were found to be repeatedly within the normal range in the majority (18 of 29; 62%) of viremic patients with or without circulating anti-HCV and were therefore not predictive of infection (Table 2). The results of RIBA II usually correlated well with those of EIA II; however, 3 of 7 patients with indeterminate results were HCV RNA positive. A detailed analysis of HCV RNA with respect to RIBA II results is shown in Table 3. Differences in
HCV VIREMIA IN DIALYSIS PATIENTS
VOL. 31, 1993 TABLE 2. Comparison of EIA II reactivity, ALT elevation, and HCV RNA status in hemodialysis patients Test and result (no. of patients)
EIA II positive (36)
EIA II negative (41)
No. of patients
HCV RNA positive 11 15 0 3
HCV RNA negative 5 5 8 30
ATsau
ALT status
Abnormal Normal Abnormal Normal
immunoglobulin levels in serum according to anti-HCV or HCV RNA status were not statistically significant. HCV genotyping. Twenty-six serum specimens positive for HCV RNA were also studied for the presence of HCV genotypes I to IV. Typing was performed as described by Okamoto et al. (18), with minor modifications (see Materials and Methods). As shown in Table 4, all four genotypes were present in hemodialysis patients, and their proportions were not significantly different from those in other patients with HCV infection (la). No correlation was found between any specific genotype and past history of blood transfusions, ALT elevations, or duration of hemodialysis treatment. However, we observed a strong trend toward a lack of immunoreactivity restricted to NS4 antigens (clOO-3, 5-1-1) both by EIA I and RIBA II in patients infected with genotype III. Thus, only 1 of 10 HCV type III-infected patients showed immunoreactivity to 5-1-1 and/or clOO-3 antigens, whereas 8 of 12 HCV type II-infected patients showed immunoreactivity. Nine of 15 EIA I-negative, EIA II-positive patients harbored HCV type III, accounting for 90% of all type III HCV isolates identified in our series. DISCUSSION Studies performed before the introduction of anti-HCV screening assays indicated that in hemodialysis patients non-A, non-B hepatitis has a mild or inapparent acute phase, but a high rate (up to 70%) of evolution to chronic liver disease, which usually follows a subclinical course (19). Almroth et al. (1) prospectively studied five patients with proven HCV infection, only one of whom had clinical signs of hepatitis, but all had a prolonged course of disease with histologically relevant lesions and low, transient antibody responses. Two studies on the prevalence of HCV viremia in hemodialysis patients performed in Taiwan (23) and Hong Kong (6) indicated that a high proportion of anti-HCVpositive patients (66 to 86%) have ongoing infection and that a variable proportion (2.5 to 12%) of HCV RNA-positive patients are seronegative. A third study from Japan (21) gave different values, reporting a low prevalence (22%) of viremia TABLE 3. Comparison of HCV RNA status with RIBA II reactivity in hemodialysis patients No. of patients
Test and result (no. of patients)
RIBA II
RIBA IT
positive
indeterminate
RIBA TI negativea
HCV RNA positive (29) HCV RNA negative (25)
23 6
3 4
3 15
a Sera from 18 of 41 EIA II-negative patients were also tested by RIBA II, confirming the negative serological test result.
2915
TABLE 4. HCV genotypes and EIA I and RIBA II reactivities for 26 HCV RNA-positive hemodialysis patients Patient no.
HCV
typ
EIA I
reactivity
5-1-1
RIBA TI reactivity c33 clOO-3
c22
1
I
-
-
-
+
+
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
TT
+ +
+
+ +
+ + + + + + +
+ + + + + + +
18 19 20 21 22 23 24 25 26
II II II II II II II II II II II III III III III III III III III III III
I-II-TII T-II-ITT
III-IV
-
-
-
+ + + + +
+ + + + +
+ + + + +
-
-
-
-
-
-
+
+
+
-
-
-
-
-
-
-
+
+
+
-
-
-
+
+
+
-
-
+
-
+
+
+
+ + + + + +
+ + + + + + + + + + + + + + + + +
+ + -
+ +
+ + + +
in anti-HCV-positive patients. Differences in the populations studied and in the procedure used for RT-PCR may explain these discrepancies. Our results are in agreement with those of others, in that (i) a high proportion of patients tested show active infection, as documented by HCV viremia, the vast majority being anti-HCV positive, (ii) a minor, but relevant proportion of anti-HCV-negative patients (7.3%) shows viremia, and (iii) ALT levels carry no predictive value of HCV infection because they proved to be repeatedly normal in 18 of 29 (62%) viremic patients. Anti-HCV positivity, as detected by EIA II and RIBA II, was the best predictor of HCV viremia in our series. EIA II appears to be more sensitive than RIBA II in detecting infected patients, since three patients that were positive by EIA II and HCV RNA were categorized as indeterminate by RIBA II, suggesting that this assay does not convincingly discriminate between productive infection or past exposure to the virus, as suggested by others (26). A plausible explanation for this phenomenon would be that immunoblotting is significantly less sensitive than ELISA in detecting low antibody titers. Ten (27.7%) of 36 anti-HCV-positive patients w ere found to be negative for HCV RNA. Serum from one of them showed decreasing optical density values in the EIA II during follow-up and was indeterminate by RIBA II, whereas the remainder showed unequivocal EIA II immunoreactivities and four of them were RIBA II indeterminate. These patients might have recovered from HCV infection or might have had low, fluctuating levels of viremia, below the limits of detection of the PCR assay; however, they were repeatedly negative on different occasions over a period of 3 months. Interestingly, six of them were HBsAg positive, and with the exception of HBsAg carriers, none of them had history of ALT elevation. No conclusion can be drawn from
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SILINI ET AL.
the observed differences in the prevalence of HCV viremia between HBsAg-positive and -negative patients because of the small number of patients; however, they are suggestive of reciprocal interactions between the two viral infections, as already pointed out by others (22). Three of 41 (7.3%) anti-HCV-negative patients were found to be positive for HCV viremia. None of them had a retrospective history of ALT elevations, in agreement with several previous surveys including our own (6, 15, 17, 23). These findings might be explained by the lack of seroconversion caused by a recent infection or an inability to mount an efficient immune response. Alternatively, some HCV strains might show significant sequence divergence from the prototype, so that seroconversion might be undetectable by current immunoassays. A mean delay time of seroconversion of 7 weeks with second-generation immunoassays (7), which may be even more prolonged in immunodeficient patients, and an estimated 5 to 10% rate of seroconversion per year of dialysis (6, 12) suggest that some of these patients might have experienced recent infections. In addition, uremic patients show a relative immunodeficiency and a reduced capacity to mount antibody responses to certain antigens such as HBsAg or tetanus toxoid (24). Moreover, immunocompetent blood donors are occasionally found to be anti-HCV negative by current immunoassays when HCV viremia titers are very low (25). With respect to this issue, it is noteworthy that all anti-HCV-negative viremic patients in our series were HCV RNA positive only after the second step of amplification, whereas all anti-HCV positive patients were positive after the first amplification step. In the study by Sheu et al. (23), patients with ALT elevations had a higher positivity rate for anti-HCV and HCV viremia than did those with normal ALT levels. In our series, a retrospective finding of ALT elevation was significantly correlated with anti-HCV positivity (16) but not with HCV viremia. Indeed, over 60% of viremic patients had normal ALT levels over a period of observation of 8 years, in agreement with findings in other risk groups (20). The result of HCV genotyping indicated that all four known common viral variants (4) coexist in our geographic area. The prevalence of different genotypes does not differ significantly from those among other anti-HCV-positive patient groups. As already suggested by others (5), there seems to be a trend toward reduced serological responses to NS4 antigens in patients with HCV type III infections, as assessed by RIBA II and EIA I. This finding argues strongly against the use of immunoreactivity patterns as indicative of infectivity or as prognostic markers of disease activity. In conclusion, a significant proportion of hemodialysis patients have ongoing HCV infection and are likely to be infectious. Serological screening is useful in identifying patients exposed to HCV; however, a significant proportion of cases eludes surveillance performed by serology alone, which not only does not predict active infection but also leads to underestimation of HCV infection. The combined figure of anti-HCV-positive and HCV RNA-positive patients points to a total proportion of exposed patients of greater than 50%. These data should prompt the use of adequate prophylactic measures to circumscribe the spread of infection within dialysis units, considering that HCV infection contributes to significant morbidity in dialysis patients and to high mortality rates in those patients that eventually undergo transplantation (2).
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