Detection of Hepatitis C Virus RNA in Hemodialysis. 1,2. Mary Kuhns,. Maria de Medina,. Anne. McNamara,. Lennox. J. Jeffers,. K. Rajender. Reddy,. Marcelo.
Detection
of Hepatitis
C Virus RNA in Hemodialysis
1,2
Mary Kuhns, Marcelo and
Maria
Silva,
Guido
de
Medina,
Carmen
Anne
McNamara,
Ortiz-lnterian,
Lennox
Margarita
J. Jeffers,
Jimenez,
Eugene
A. McNamara,
Abbott
Laboratories,
Soc.
Nephrol.
1994;
and/or
additional
Abbott
M. de Medina, L.J. Jeffers, K. P. Reddy, M. Silva, C. Ortiz-Interian, M. Jimenez, E. P. Schiff, G. Perez, Department of Medicine, University of Miami School of Medicine and Veterans Administration Medical Center, Miami, FL (J. Am.
HCV-positive
infectious. negative infection the
4:1491-1497)
clinical
significance
antibodies
remains
tionship
was
45 mo
prevalence in dialysis
to assess and
undergoing
dialysis were evaluated 1990. The mean duration alysis
high (HCV)
In order
seropositivity
patients
63
C virus
undefined.
between
tivity,
of the
to hepatitis
the rela-
potential
infec-
maintenance
hemo-
between April of maintenance
(range,
13 to
144).
of pa-
and May hemodi-
Eighty-two
per-
cent (52 of 63) had received blood transfusions, and 16% (10 of 63) had a history of iv drug abuse. Serum samples were analyzed by HCV-cDNA polymerase chain reaction; antibodies to HCV structural (core) and nonstructural regions NS3 and NS4 were determined by enzyme immunoassay. Specimens repeatedly reactive for anti-HCV and HCV-RNA-positive samples were tested by HCV MATRIX@ dot immunoblot assay and HBV-DNA PCR. Twenty-five percent (16 of 63) were anti-HCV-positive. Of the 16 anti-HCVpositive patients, HCV-RNA was detected in 5(31%) with
the
NS3 primers
and
in 12 (75%)
with
5’-noncod-
ing primers. Among the anti-HCV-negative patients, HCV-RNA was detected in 2 (4.3%) of 47 patients. Eleven
of the
, ReceIved
April 13, 1993.
2
Presented
Nephrology,
I 8 patients
In part
Accepted
at the
Baltimore.
24th
MD.
with
HCV
infection
(anti-
July 26, 1993.
Annual
November.
Meeting 17-20,
of the 1991
Society
American (J
Am
Soc
Nephrol
1991:2:334). Correspondence to Dr. GO. Perez, Dialysis Unit (1 1 1-C), veterans Administration Medical Center, 1201 NW 16 Street, Room A-1005, Miami, FL 33125. 3
1046-6673/0407-1491503.00/0
Journal Copyright
Journal
of the American C 1994
of the
by
the
Society American
American
of Nephrology society of Nephrology
Society
of Nephrology
of
A
patients,
or
past
had viral
virus and/or hepatitis is present in at least suggesting
evidence
infections
that
of
(human
B virus). In 75% of antithey
may
be
The detection of HCV-RNA in anti-HCVpatients may indicate early or chronic HCV not detected by current antibody assays or
inability
hepatitis
ABSTRACT
HCV-RNA-positive)
immunodeficiency summary, HCV-RNA
of these
antibody
Key Words:
tients
R. Schiff,
present
significant
The
Reddy,
Perez3
HCV M. Kuhns, Park, IL
K. Rajender
patients
to mount
or sustain
a
response.
Hemodialysis, hepatitis B virus DNA, polymerase
C, hepatitis chain reaction
C virus RNA,
high virus
prevalence of antibodies to the hepatitis C (anti-HCV) has been demonstrated among high-risk populations. including patients on maintenance hemodialysis ( 1 -5). In our previous study, 12% of 90 patients and none of the staff were positive for anti-HCV by first-generation screening assays (6). Similarly, from 5.4 to 1 2% of 387 dialysis patients on the “Eurotranspbant” waiting list were antibodypositive (7). However, antibody assays cannot estabbish whether an anti-HCV-positive patient has recovered from the infection or remains a potentially infectious carrier of the virus (8- 1 2). The amplification of nucleic acid sequences by pobymerase chain reaction (PCR) is a sensitive and specific technique to detect viremia. Anti-HCV-positive patients may lack detectable serum HCV-RNA because of intermittent viremia, infection with a strain of HCV not recognized by the PCR primers, or the cessation of active viral replication in recovering individuals with residual antibody reactivity. In contrast, seronegative individuals may be HCV-RNA-positive during early acute disease because HCV antibodies often appear weeks after the onset of hepatitis (1 3). Moreover. the persistence of an active HCV infection may not abways be associated with sustained antibody reactivity because of host immune response factors. The prevalence of antibody to the hepatitis B core antigen (anti-HBC) is high (54 to 73%) among antiHCV-positive blood donors and hemodialysis patients (6, 1 3). Studies of hepatitis B virus (HBV) and HCV interaction in individuals with dual infections have suggested that HCV may suppress or terminate the
1491
HCV RNA in Hemodialysis
Patients
hepatitis B surface antigen (HB8Ag) carrier state. In one longitudinal study, four of five HBsAg-positive and anti-HCV-posltive patients who later cleared serum HBsAg continued to have detectable serum levels of HBV-DNA by PCR (14). Cases of potentially infectious HB5Ag-negative HBV, as determined by the detection of serum HBV-DNA, have been described in individuals with and without anti-HBc (15-17). In order to assess the relationship between HCV seropositivity and potential infectivity. we analyzed serum samples from patients undergoing maintenance hemodialysis by second generation, anti-HCV enzyme immunoassay (EIA) supplemental anti-HCV assays (HCV MATRIX#{176}; Abbott Laboratories, Abbott Park, IL), and HCV-cDNA PCR using primers from the NS3 and 5’-noncoding regions of the HCV genome. In addition, we examined HCV-positive serum samples for potential concurrent hepatitis B viremia by PCR for HBV-DNA.
METHODS Patients Sixty-three patients undergoing maintenance hemodiabysis at the Miami Veterans Affairs Medical Center and the University of Miami/Jackson Memorial Medical Center were evaluated between April and May 1990. Blood samples were analyzed under code. Routine hemodialysis techniques were used, with 4h treatments performed thrice weekly and with cellubosic membranes with acetate or bicarbonate solutions of standard composition. Artificial kidneys were not reused, and universal blood precautions have been in practice since 1988. The mean age of the patients (49 men and 14 women) was 55 yr (range. 29 to 77 yr). The mean duration of maintenance hemodialysis was 45 mo (range. 13 to 144 mo). Transfusion records indicated that 52 (82.5%) of the patients had received blood transfusions (mean, 19.5; range, 0 to 204 U). Sixteen percent (1 0 of 63) had a history of iv drug abuse (IVDA).
Serological
Testing
Antibodies to HCV putative structural (core) and nonstructural regions NS3 and NS4 were determined by HCV EIA 2.0 (Abbott Laboratories). Repeatedly reactive specimens and HCV-RNA-positive samples were tested by HCV MATRIX#{176}, a semiautomated dot immunobbot assay consisting of an array of HCV recombinant polypeptides individually spotted on nitrocellulose. Antigen test panels consisted of NS3, NS4, and core polypeptides (18). HBsAg and anti-HBc were determined by RIA (Abbott Laboratories).
1492
HCV-RNA
cDNA
PCR
Serum samples were stored at -20#{176}C until use. Twenty-five microliters of serum was diluted 1 0-fold in digestion buffer and incubated for 60 mm at 30#{176}C. The final concentration in the diluted sample was 50.5 mM in Tris-HC1 (pH 8.0). 1 mM EDTA (pH 8.0), 0. 1 M NaCb, 0.5% sodium dodecyl sulfate (SDS), 1 mgjmL of proteinase K, and 20.2 g/mL of tRNA. The solubilized sample was then sequentially extracted with phenol. phenol-chloroform/isoamyl alcohol, and chboroform/isoamyl alcohol. Nucleic acids were precipitated overnight at -20#{176}Cby the addition of vol of 2 M sodium acetate and 2.5 vol of cold ethanol. The precipitate was recovered by centrifugation and resuspended in 5 L of diethylpyrocarbonate-treated water containing 1 U/L of RNasin and 1 mM d!thiothreltol. The RNA was heated at 37#{176}C for 30 mm and 65#{176}C for 3 mm and then cooled on ice. cDNA was synthesized at 37#{176}C for 1 h in a final volume of 25 iL in 100 mM Tris (pH 8.3), 76 mM KC1, 10 mM MgCb2, 10 mM dithiothreitol, 1 mM each dATP, dGTP, and dTTP, 0.5 mM CTP, 4 M each primer, 5 U of avian myeboblastosis virus (AMV) reverse transcriptase, and 25 U of RNasin. The reaction mixture was then diluted with 50 jL of water, heated in a boiling water bath for 10 mm, and placed on ice. PCR (50 cycles for NS3 primers, 35 cycles for 5’noncoding region primers) was performed in a final volume of 100 L containing 32.5 mM Tris (pH 8.3), 56.5 M KC1. 3.6 mM MgCl2, 0.34 mM each dATP, dGTP, and dTTP, 0.22 mM dCTP, 1 jzM each primer, and 5 U of Taq polymerase. Initial denaturatlon was 5 mm at 94#{176}C (subsequent cycles were 1 mm). Annealing was 2 mm at 37#{176}C,and extension was 3 mm at 72#{176}C (10 mm in last cycle). For each PCR assay. multiple negative and positive control serum samples were extracted and amplified. Reverse transcriptasePCR reagent controls were also included in each assay. HCV-cDNA PCR products from the 5’-noncoding region were detected by spot and solution hybridization with a (32Pjoligomer probe specific for the region between the primers. For spot hybridization, PCR products were denatured in 0.35 M NaOH and 0.0 1% SDS for 1 0 mm and 5 L was then spotted directly on a nylon membrane and neutralized with 0.5 M Tris-3.0 M NaCl (pH 7.4). While damp, the filters were exposed to ultraviolet radiation to cross-link the DNA to the filter and then baked at 80#{176}C.Prehybridization (1 h) and hybridization (2 h) were performed at 65#{176}C in 10% dextran sulfate, 6 x sodium chboride/ sodium citrate buffer (SSC). Filters were washed twice for 5 mm at room temperature in 2 x SSC0. 1% SDS and twice for 15 mm at 65#{176}C in 0. 1% SSC0. 1 % SDS. Autoradiography results were scored under code. HCV-cDNA PCR products from the 5’-noncoding 1/
Volume
4
.
Number
7
1994
#{149}
Kuhns
region were also detected by quantitative solution hybridization by modification of a method used for direct detection of DNA and PCR products (19,20). Twenty microliters of PCR products was brought to a final volume of 200 L by the addition of 30 L of autoclaved water, 50 iL of 0.3 M NaC1, and 100 L of 2 x tryptone yeast medium. The remaining steps of the protocol were a modification of the Abbott assay for hepatitis B viral DNA in serum (Abbott Laboratories)(19). The diluted sample was solubilized by the addition of 1 0 tL of proteinase K followed by 20 L of 1 . 1 5 N NaOH-0.74 M NaCl. Solution hybridization was carried out by the addition of [32Pjoligomer probe (specific for the amplified HCV sequence) in 70 L of 0.55% SDS, 0.68 M sodium phosphate buffer (pH 6.3). and 0. 193 M NaCl for 2 h at 65#{176}C. Radioactive hybrids were separated from free probe by the passage of the 300-L hybridization reaction over a 3.4-mL sepharose CL6B (Pharmacia, Sweden) chromatography column. Radioactive hybrids in a total elution volume of 1 ,800 L were then quantitated by scintillation counting. A cpm value at least 2 SD from the negative control mean was considered positive. HCV-cDNA PCR products from the NS3 region were detected by quantitative solution hybridization, as described above, with a single-stranded [‘25IJDNA probe and sepharose CL4B chromatography (hybrids eluted in a total volume of 1 ,750 ML). Results were confirmed by spot hybridization, as described above, with a single-stranded (32PJDNA probe. The sensitivity of the HCV-cDNA PCR was evaluated with chimpanzee plasma of known hepatitis C chimpanzee infectious dose (CID). For 5’-noncoding primers, one CID/PCR was detected; for NS3 primers, 5 CID/PCR were detected. Figure 1 shows examples of solution hybridization, spot hybridization, and Southern blot results after PCR (5’-noncoding region) of an HCV-RNA-positive human control serum sample. The anti-HCV-positive control was previously found by cDNA-PCR to be equivalent to a chimpanzee plasma sample containing 2 x 1 o4 CID/mL. The extraction and amplification of 25 iL of 1 : 1 60 dilution thus represented 3 CID per PCR. In other experiments, Southern blot analysis of NS3 PCR products of negative and positive controls and selected specimens demonstrated a single band of the expected size (data not shown). The specificity of HCV-cDNA PCR was also evaluated by testing normal human serum specimens (N = 34 for NS3 primers, N = 50 for 5’-noncoding primers). All normal serum samples were negative for HCV-RNA.
Oligonucleotide and Probes Conserved
Journal
of the
Sequences HCV
American
5’-noncoding
Society
of HCV region
of Nephrology
Primers primers
(293-
+
+
++
++
10000
++
+++
+++
et al
A
C
B
----C_ C
1000
C,
a. 0
100
C
10
S
I, NEG.
1:200
1:160
POSITIVE
1:100
1:50
CONTROL
1:20
1:10
1.5
1.2
DILUTION
Figure 1. Detection of HCV 5’-noncoding region cDNA PCR products in positive and negative controls. PCR products were generated with primers from the 5’ -noncoding region and analyzed by spot hybridization, solution hybridization, and Southern blot. A (32P)oligomer probe and high-stringency conditions were used in all three hybridization methods. A dilution series of the positive control was assayed by (A) spot hybridization (5-,I aliquot of PCR product) and by (B) solution hybridization (20-SI aliquot of PCR product). Each solution hybridization point represents an independent extraction and amplification. The Southern blot (C) shows results for the negative (NEG.) control sample and a 1:5 dilution of the positive (P05.) control. A single band of
approximately
300 base
pairs (arrow)
was observed.
base-pair PCR product) were 5’-CAC TCG CAA GCA CCA TAG ATC ACT CCC CTG TGA. The probe Sequence was 5’-CTG CGG AAC CGG TGA GTA CAC CGG AAT TGC (21). NS3 primers (586-base-pair PCR product) were 5’GCA TGT CAT GAT GTAT and 5’-ACA ATA CGT GTG TCA C (8). A single-stranded probe (average length of 250 bases) for the detection of NS3 PCR products was synthesized by primer extension (primer sequence 5’-CAG TCT GTA TAG CAG G) in the presence of limiting concentrations of [‘25IJdCTP or [32P]dCTP. followed by purification on an alkaline gel sizing column.
HBV-DNA
PCR
HBV-DNA PCR with primers from the surface gene region was performed as previously described (20). Briefly, 50 L of serum was solubilized for 2 h at 37#{176}C by the addition of an equal volume of buffer containing 29.7 M Tris-HC1 (pH 8.0), 29.7 mM EDTA (pH 8.0), 0.44 M NaCl, 5.9 mg/mL of proteinase K, and 5.9% SDS. Samples were then extracted with
1493
HCV
RNA in Hemodialysis
Patients
phenol and chloroform/isoamyl alcohol. DNA was precipitated overnight at -20#{176}C in the presence of sodium acetate and ethanol. HBV surface gene primers (5’-CTC T’fl GTT TTG TFA GGG TTT AAA T and 5’-CTC CCT TAT TCG TCA ATC TFC TCG AGG ATT) amplified a 750-base-pair segment. Surface gene PCR was performed in a 1 00-zl volume containing 50 mM KC1, 10 mM Tris-HC1 (pH 8.3), 10 mM MgC12, 0.00 1 % gelatin, dNTP (0.2 mM each of dATP, dGTP, dTTP, dCTP), 1 M in each primer, and 5 U of Taq polymerase with 25 cycles of amplification (1 mm, 94#{176}C;2 mm, 37#{176}C;5 mm, 72#{176}C).The initial denaturation was for 2.5 mm, and the final extension was for 10 mm. Negative and positive control samples were included in each PCR. Positive control HBVDNA standards containing known concentrations of HBV-DNA were prepared by the dilution of a previously quantitated native hepatitis B viral concentrate. PCR products from the HBV surface gene region (25-L aliquot) were quantitated by solution hybridization, as described above, with a single-stranded (‘251]DNA probe specific for the HBV region between the primers in a modification of the commercially available Abbott assay for hepatitis B viral DNA (19,20). SensitivIty was one HBV-DNA copy per PCR.
TABLE I
.
Summary No. of Blood Transfusions
Patient
No.
3 6 7 lob 20 26 29 35
15 0 35 3 9 28 4 7
36
7
37 39 45
0 24 18 I
47b
55
20 20
56d 59 63
74 26 123
50C
of data
on patients
positive
This sensitivity was equivalent to 20 copies/mL when 50 L of serum were extracted and amplified. Samples for HCV and HBV PCR were prepared with disposable sterile pipettes in an area free of other clinical samples or PCR products. The potential for cross-contamination was also controlled by the use of aerosol barriers and frequent glove changes. Precautions were taken to avoid PCR contamination according to the principles outlined by Kwok and Higuchi (22). Multiple freeze-thaw cycles were avoided whenever possible for specimens used in HCV-cDNA PCR testing.
RESULTS Serum samples were evaluated by
from 63 hemodialysis patients second-generation HCV EIA and by HCV-cDNA PCR with primers from the NS3 and conserved 5’-noncoding regions of the HCV genome. Serologic and virobogic data for patients positive for HCV-RNA or anti-HCV are summarized in Table 1. Twenty-five percent (1 6 of 63) of the patients were anti-HCV-positive. Thirteen were reactive for three gene products (NS3, NS4, core), and three were p051tive for anti-N53 and anti-core. Of the 1 6 anti-HCV-positive patients, HCV-RNA
for anti-HCV
or HCV-RNA#{176}
HCV MATRIX#{174} IVDA
Anti-HIV
Anti-HCV-2
-
+
-
+
+
+
+
+
-
+
-
+
+
+
+
+
-
-
-
+
-
+
-
-
-
+
-
+
-
+
+
+
+
+
+
+
Anti-NS4
Anti-NS3
Anti-core
12.299 18.909 11.675 14.851 12.188 0.452 17.740 12.708
5.386 18.260 6.114 17.351 11.734 16.961 19.117 12.916
21.299 20.006 20.357 14.935 16.117 4.401 20.331 20.110
0.100
0.100
0.400
0.873 7.500 0.000 18.013 11.403 0.538 9.377 9.000 14.500
19.481 19.500 0.000 17.760 15.968 1.210 5.846 16.500 13.100
20.584 11.800 0.237 18.442 16.214 16.747 16.818 12.800 14.000
Anti-HBc
HCV-PCR
HCV-PCR
(NS3)
(5’NC)
-
-
+
+
-
-
-
-
+
-
+
-
-
+
-
+
+
-
-
-
-
+
-
-
-
-
-
-
+
-
-
+
-
+
-
-
+
-
+
-
-
+
-
+
+
+
-
-
-
+
+
-
-
+
-
+
-
-
+
-
+
-
-
-
-
-
-
-
+
-
+
-
-
+
-
-
+
+
+
+
+
+
-
-
+
+
-
+,
+,
-
two
Independent
b
Elevated
C
Partner
of IVDA.
d
Patient
was also HBsAg-posltive.
1494
alanine
extractions
aminotransferase
and
level.
-,
+
Anti-NS4. anti-NS3. and anti-core were determined by HCV MATRIX#{176} dot immunoblot assay (assay values for HCV MATRIX-posltive from I to 25); HCV c-DNA PCP results are shown for two regions: NS3 and the 5’-noncoding region (5’NC); HCV-cDNA PCP (5’NC) results are shown for three serial samples from Patient No. 36. All three samples were anti-HCV-negative. Positive PCI? results were a
at least
HBV-PCR
+,
samples and
-
range
HBV
reproduced
PcI?
in
amplifications.
Volume
4
Number
#{149}
7
‘
1994
Kuhns
was detected in 5 (3 1 %) with the N53 primers and in 12 (75%) with the 5’-nocoding region primers. All samples positive with the NS3 primers were also positive with the 5’-noncoding primers. HCV-RNA was also detected in 2 (4.3%) of the 47 patients who were negative for antibodies to all HCV antigens tested. For one of these patients (Table 1 ; Patient No. 36), additional samples were obtained at 1 and 1.5 yr after the initial testing. Both samples were HCVRNA-positive but remained anti-HCV-negative. Twelve of the 1 8 patients with HCV infection, as demonstrated by HCV antibodies or RNA, had evidence of additional present or past viral infections. Ten were anti-HBc-positive, three were HBV-DNApositive (only one patient was HB5Ag-positive), and three were anti-HIV-positive. Results for one patient (Table 1 , Patient No. 59) indicated coinfections with HCV, human immunodeficiency virus type 1 , and HBV. Risk factors associated with the 1 8 patients positive for HCV markers were a history of drug abuse in 7, a sexual partner with IVDA history in 1 , and transfusions (mean, 26 U; range, 1 to 123) in 16 patients (Table 1 ). The mean duration of dialysis was 42 mo (range, 3 to 144 mo). Alanine aminotransferase values were elevated in only two patients. All patients were asymptomatic without clinical evidence of chronic liver disease. Both patients who were HCV-RNA-positive but anti-HCV-negative had no history of IVDA but had multiple transfusions. Of the 1 2 individuals with evidence of multiple infections (1 0 anti-HBc-positive. 3 anti-human immunodeficiency virus-positive), 6 had a history of IVDA and 1 1 had received transfusions (mean, 1 9 U; range, 1 to 74). For comparison, a history of IVDA was noted for 3 of the 45 subjects negative for HCV markers. Forty (89%) of 45 patients had a history of transfusion (mean, 21 U; range, 0 to 204), and 16 (36%) were anti-HBc-positive. The mean time on dialysis for this group was 24 mo (range, 3 to 144 mo).
DISCUSSION HCV infection has become the most common type of hepatitis among patients on maintenance hemodialysis (23). In our previous study, using first-generation EIA for the detection of antibody to c 100-3 (NS3/NS4 regions), we found a prevalence rate of 12% (6). Second-generation EIA, which detects antibodies to antigens encoded by the core, NS3, and NS4 regions, has been shown to identify up to 25% more patients in high-risk populations (24). HCV EIA 2.0 results in this study demonstrated a prevalence rate of 25%, similar to that reported by other centers (1 .5,7,25). Supplemental testing consisted of dot immunoblot assays for HCV antigens (HCV MATRIX#{174}), including one putative structural (core) and two non-
Journal
of the
American
Society
of Nephrology
et al
structural (NS3 and NS4) antigens currently included in the second-generation screening assays. All of the anti-HCV-positive patients displayed antibodies to multiple independent HCV antigens. Most HCV-infected individuals are chronic viremic carriers, as determined by PCR (26,27). In this study, HCV-RNA was detected in 75% of anti-HCV-positive patients. The detection of HCV-RNA in the sera of anti-HCV-positive patients was markedly greater with primers from the highly conserved 5’-noncoding region than with primers from the more variable NS3 region (75 versus 3 1 %). HCV-RNA-positive individuals are potentially infectious and at risk for the development of chronic liver disease (9- 1 1 ,26); HCVinfected dialysis patients may also be susceptible to the same risks. Two recent studies (27,28) have measured HCV-RNA by PCR in hemodialysis studies. Those studies were performed in different patient populations, and the authors found a higher prevalence of alanine aminotransferase elevation than that found in our study (27,28). HCV-RNA was also detected in two anti-HCV-negative patients, whereas no PCR positives were observed among the healthy control population. The data are consistent with a recent study from Japan in which HCV-RNA was detected in 1 3 (8 1 %) of 16 anti-HCV-posltive patients. as well as in 7 of 1 4 antiHCV-negative patients with chronic liver disease (1 0). Anti-HCV-negative, HCV-RNA-positive patients are often immunocompromised (e.g. . transplant and dialysis patients and those with acquired immunodeficiency syndrome). Of interest, in this study. both patients who were HCV-RNA-positive and anti-HCVnegative. gave no history of IVDA. Our results also suggest that screening tests may underestimate the prevalence of current HCV Infection in dialysis patients. The presence of serum HCV-RNA in seronegative patients may indicate early or chronic HCV infection not detected by current antibody assays. Alternatively, this phenomenon may be associated with the inability of immunodeficient uremic patients to mount or sustain a significant antibody response. PCR for HBV-DNA revealed current coinfection with HBV and HCV in two HBsAg-negative patients. One patient (Table 1 ; Patient No. 59) was positive for anti-HCV, HCV-RNA, and anti-HBc. The second patient (Table 1 ; Patient No. 36) was HCV-RNA-positlve but had no additional HBV or HCV markers. HBsAgnegative HBV infection, as determined by the presence of HBV-DNA, has been described (1 5- 1 7). In one report, a patient superinfected with HCV lost HBSAg but continued to be serum HBV-DNA-positive by PCR and, therefore, potentially infectious (14). Thus, concurrent infection with HBV and HCV may complicate the diagnosis of chronic liver disease. HBV-DNA can be detected by PCR in HB5Ag-negative
1495
HCV RNA in Hemodialysis
Patients
patients, even in the absence of other serologic markers for HBV. such as anti-HBc and anti-HBs, but more often in the presence of these markers. The frequency of these findings and the relative infectivity of these patients have not been characterized in the dialysis population. Testing for viral nucleic acid may be necessary to adequately assess the status of chronic HBV and HCV infection in clinical studies in dialysis patients. The current guidelines for the prevention of nonA, non-B hepatitis in dialysis units advocate the appbication of Universal Precautions and scrupulous aseptic techniques (29). Specific recommendations from the Centers for Disease Control and Prevention regarding surveillance and prevention of HCV infection in hemodialysis units have not been published. Recent reports of hemodialysis-associated hepatitis (30-34), as well as the results of this study, provide additional information that may help formulate specific infection control measures for HCV in hemodialysis units.
ACKNOWLEDGMENTS The
authors
thank
Ana
M. Orti.z
for
secretarial
assistance.
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