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Blood, Vol. 94 No. 4 (August 15), 1999:
pp. 1183-1191
Low Levels of Hepatitis C Virus RNA in Serum, Plasma, and Peripheral
Blood Mononuclear Cells of Injecting Drug Users During Long
Antibody-Undetectable Periods Before Seroconversion
By
Marcel Beld,
Maarten Penning,
Marieke van Putten,
Anneke van den
Hoek,
Marjolein Damen,
Michèl R. Klein, and
Jaap Goudsmit
From the Academic Medical Centre, University of Amsterdam, Department
of Human Retrovirology, Amsterdam, The Netherlands; the Municipal
Health Service, Department of Public Health and Environment, Amsterdam,
The Netherlands; the Central Laboratory of The Netherlands Red Cross
Blood Transfusion Service, Amsterdam, The Netherlands; and
the Medical Research Council (MRC) Laboratories, Banjul, The Gambia.
 |
ABSTRACT |
Screening of antibodies to hepatitis C virus (HCV) is widely used
for monitoring the prevalence of HCV infections and to assess HCV
infectivity. Among HCV-infected individuals in the general population,
the interval between the detection of HCV RNA and the development of
HCV antibodies is usually 5 to 6 weeks, but in rare cases,
seroconversion may be prolonged up to 6 to 9 months. In this study, we
tested for the presence of HCV RNA during the antibody-undetectable
period of 19 drug-injecting HCV seroconverters to gain insight into the
antibody-negative carrier status in this population. HCV seroconversion
status was determined by testing the first and last serum samples
obtained from each subject, using third-generation antibody screening
and confirmation assays. Serial samples were tested for HCV-specific
antibodies to establish the moment of seroconversion and HCV RNA by
single reverse transcriptase-polymerase chain reaction (RT-PCR) and
branched DNA assay (bDNA) in serum. Plasma and peripheral blood
mononuclear cells (PBMCs) were independently collected and tested for
HCV RNA. HCV RNA-positivity was confirmed by Southern blot
hybridization and sequencing of serial samples. The 19 HCV
seroconverters had a mean follow-up of 5 years (range, 1 to 8 years).
Of the 19, 4 were human immunodeficiency virus (HIV)-infected before
HCV seroconversion. HCV RNA was detected in serum before seroconversion
in 12 (63.2%) of the 19 HCV seroconverters, independent of HIV status.
In 7 of these 12, the antibody-undetectable period was relatively short
(2 to 10 months). The other 5, who were all HIV-negative before HCV
seroconversion, had intermittent low levels of HCV RNA before
seroconversion for a period of more than 12 months, with a mean of 40.8 months (range, 13 to 94 months). In all 5 individuals, independent
repeats of the experiments confirmed the presence of HCV RNA in serum,
and in 3 of these individuals, HCV-positivity was confirmed in
independently collected plasma and PBMC samples. Low levels of HCV RNA
may be present during prolonged antibody-undetectable periods before
seroconversion in a number of injecting drug users. Independent of HIV
status, their immune system appears to be unable to respond to these
low HCV RNA levels and was sometimes only activated after reinfections with distinct HCV genotypes. These results indicate that primary HCV
infection may not always elicit the rapid emergence of HCV antibodies
and suggests that persistent low levels of HCV RNA (regardless of the
genotype) may not elicit at all or delay antibody responses for
prolonged periods of time.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HEPATITIS C VIRUS (HCV), an RNA virus
with marked genetic heterogeneity, is the etiological agent of most
cases of posttransfusion and community-acquired non-A and non-B
hepatitis.1 This blood-borne virus is widespread among
injecting drug users (IDUs),2-4 and HCV infections may
cause a benign, asymptomatic disorder with an indolent
course5 but can eventually cause progressive liver disease,
cirrhosis, and liver cancer.6-8 The illness has a complex
course, with RNA levels that are often transient.9-12 Antibodies to HCV are detected by immunoassays, and their presence is
closely related with infectivity, especially in blood
donors,13-15 hemodialysis patients,16
hemophiliacs,17 and patients with chronic
HCV.18,19 Current immunoassays detect most individuals with
HCV infections, and HCV RNA has been detected during short antibody-undetectable periods (the window phase from infection to the
development of antibodies to HCV) in persons infected by blood
transfusions20,21 or surgical procedures22 and
in experimentally infected primates.23,24 However, up to
half of immunosuppressed patients, such as organ transplant patients,
fail to generate antibodies to HCV,25 and IDUs also have a
high proportion of delayed antibody responses.2 In 2 other
studies, HCV RNA was significantly more detectable in whole blood than
in plasma samples among antibody-negative individuals, indicating that
a proportion of HCV RNA in peripheral blood might be
missed.26,27 Unexpected clinical profiles, with HCV RNA
present for a period of 5 years without antibodies to HCV, were
observed in experimentally inoculated chimpanzees.28
Spontaneous loss of antibodies to HCV, or seroreversion, among
immunocompetent individuals has recently been described,29 and we have observed a complete loss of antibodies to HCV in
individuals both with and without reinfection.30
Therefore, the present study was performed to identify and
confirm the presence of HCV RNA in serum and other blood compartments
before the moment of seroconversion in the high-risk group of IDUs.
| |
PATIENTS AND METHODS |
Patients and sample collection.
The study population consists of IDUs participating in the Amsterdam
Cohort Studies on human immunodeficiency virus (HIV) and acquired
immunodeficiency syndrome (AIDS) among IDUs. From a cohort started in
December 1985,31 we selected drug users in March 1996 who
were observed for at least 3 years and had at least 7 visits (n = 358).
The 19 HCV seroconverters (10 women and 9 men; mean age, 38 years) and
the cohort of 358 participants (148 women and 210 men; mean age, 42 years) from which they are recruited are representative of the original
cohort and are 99% whites. A previous study3 identified
risk factors for a prevalent HCV infection, which included a history of
injecting drug use and duration and frequency of injecting drug use.
Because our aim was to study incident cases of HCV infection, we
therefore selected in March 1996 only drug users with a history of
injecting drug use who had sufficient follow-up (>3 years) and study
visits (>6). Because of this selection, we included in this study
mainly drug users with a relatively long career of injecting drugs, of whom 88.3% (n = 316) were found to be already infected with HCV at
intake. Among the remainder of 42 participants, 23 remained seronegative throughout the study period, whereas 19 seroconverters were identified. Their HCV seroconversion status was established by
screening the first and the last serum samples for HCV antibodies, and
serial samples of the seroconverters were then tested to establish the
approximate seroconversion point. The date of HCV seroconversion was
determined by calculating the midpoint between the last seronegative and first seropositive sample. Nineteen HCV seroconverters, of which 1 reseroconverted, were identified and studied longitudinally for the
presence of HCV RNA. Serum samples were initially used to determine the
presence of HCV RNA before seroconversion. The findings of HCV
RNA-positive samples before HCV seroconversion were confirmed by
detecting HCV RNA in serum and peripheral blood mononuclear cells
(PBMCs) or plasma sampled at the same time point. In addition, the
branched DNA (bDNA) assay was performed on the same serial serum
samples before and after HCV seroconversion. Serum and EDTA-blood
samples of the 19 HCV seroconverters were drawn at different Municipal
Health stations in Amsterdam. For final handling and storage, all serum
samples were sent to the Academic Medical Centre, whereas all
EDTA-blood samples were sent to the Central Laboratory of The
Netherlands Red Cross Blood Transfusion Service. Serum samples were
stored initially at +4°C, centrifuged, aliquoted, frozen at
 20°C within 24 hours of collection, and ultimately stored at
70°C. PBMCs and plasma samples, also initially stored at
+4°C, were separated from EDTA-blood by density-gradient centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Cryopreservation on aliquoted PBMCs was performed using
computer-automated freezing in liquid nitrogen.
Serological tests.
Samples were tested for the presence of antibodies to HCV by the
third-generation enzyme immunoassay (EIA 3.0; Abbott Laboratories, Chicago, IL). All positive EIA 3.0 assays were confirmed by the third-generation strip immunoblot assay (SIA, RIBA; Chiron Corp, Emeryville, CA). Antibodies to HIV-1 were determined with commercial EIA (Abbott Laboratories) and confirmed by Western blot (Diagnostic Biotechnology, Herent, Belgium). Individuals who remained HIV-negative were screened on all consecutive samples by EIA. All serological assays
were performed according to the manufacturer's manual.
Detection of HCV RNA by reverse transcriptase-polymerase chain
reaction (RT-PCR) in serum and plasma samples.
HCV RNA was isolated from a 100-µL serum or plasma sample, according
to Boom et al,32 and was used immediately in single RT-PCR
experiments, using primers located in the 5' noncoding region, or
stored at 70°C as previously described.12
Briefly, one fifth (10 µL) of the isolated RNA was subjected to
reverse transcription and a single PCR. For reverse transcription, 10 µL RNA was incubated with 25 ng of antisense primer (nt 319-324) 5'-ACCTCC-3' for 5 minutes at 65°C and then cooled down
to 42°C. Finally, 14 µL of the reverse transcription mixture was
added, containing 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 0.1%
Triton X-100 (Packard Instrument Co, Inc, Downers Grove, IL), 6 mmol/L MgCl2, 0.6 mmol/L of each dNTP, 20 U RNAse inhibitor
(Promega, Madison, WI), and 100 U SuperScript II (Life Technologies,
Gaithersburg, MD). The mixture was incubated for 30 minutes at
42°C, and 12.5 µL was used for the single PCR in duplicate. For
the PCR, the GeneAmp PCR carry-over prevention kit (Perkin Elmer Cetus,
Branchburg, NJ) was used to avoid contamination. The PCR was performed
in a 50 µL volume containing 100 ng of sense primer (nt 47-68)
5'-GTGAGGAACTACTGTCTTCACG-3', 100 ng of antisense primer
(nt 292-312) 5'-ACTCGCAAGCACCCTATCAGG-3', 2.5 U Ampli-Taq
polymerase (Perkin Elmer Cetus), 50 mmol/L Tris-HCl, pH 8.3, 20 mmol/L
KCl, 1.2 mmol/L MgCl 2, 2.5 µmol/L of each dNTP, 25 µmol/L dUTP, and 0.5 U Uracil N-glycosylase (Perkin Elmer Cetus). The
thermal cycler (type 480; Perkin Elmer Cetus) was programmed as
follows: 5 minutes at 95°C and subsequently 40 cycles of 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes, and
then incubation of samples for 8 minutes at 72°C. PCR products were subjected to electrophoresis in 2% agarose containing ethidiumbromide and visualized under UV light. As positive controls, we used a pool of
HCV-positive serum quantified by the bDNA technology (Chiron Corp) to a
level of 1.6 × 106 HCV RNA copies/mL and 100-fold
dilution. The sensitivity of our single RT-PCR was evaluated by serial
2-fold dilutions of the quantified pool of serum and was found to have
a detection limit of approximately 103 HCV RNA copies/mL
(results not shown). As negative controls, we used a pool of
commercially available serum (seronegative for HIV, HBV, and HCV) and
TE (Tris/EDTA). All positive and negative controls were tested in
parallel with the test samples throughout the entire procedure,
starting from RNA extraction. Serum samples yielded no different PCR
results from plasma samples (results not shown). The RT-PCR rendered
good duplicates unless its detection limit was reached, in which case
it rendered a plus/minus duplicate (Poisson distribution). Samples were
randomly tested for the presence of HCV RNA, and all precautions were
taken to avoid any possible contamination, using separated locations
for extraction of specimen, amplification of HCV RNA, and analyses of
PCR products.
Isolation and detection of HCV RNA by RT-PCR in PBMCs.
HCV RNA was isolated from 100 µL PBMCs containing approximately
105 cells/mL using Total RNA Isolation Reagent (TRIzol
Reagent; Life Technology), which is designed for the isolation of total
RNA from cells and tissue, according to the manufacturer's manual. HCV
RNA was detected in PBMCs with nested PCR, processing one tenth of the
initial RT-PCR products under the same conditions as in the single PCR,
but with 25 cycles using sense primer (nt 74-91)
5'-AGCGCCTAGCCATGGCGT-3' and antisense primer (nt 243-260) 5'-TACCACAAGGCCTTTCGC-3', which were extended at the
5'-end with the 21 M13 primer and the reverse M13 primer,
respectively. Samples were randomly tested for the presence of HCV RNA,
and all precautions were taken to avoid any possible contamination,
using separated locations for extraction of specimen, amplification of
HCV RNA, and analyses of PCR products.
HCV RNA quantification.
The HCV RNA load in serum was determined longitudinally by the bDNA
signal amplification assay 2.0 (Quantiplex HCV RNA; Chiron Corp)
according to the manufacturer's manual. All samples were tested in
duplicate, and the mean value of the duplicate tests was used for data
analysis. Viral load, expressed as HCV RNA copies per milliliter, was
determined by comparison with an external standard curve, having a
quantitation limit of 2.0 × 105 HCV RNA copies/mL.
Specificity and confirmation of HCV PCR products.
The specificity of the PCR products was confirmed by using a
5'-digoxigenine labeled probe33 (nt 264-291),
5'-TTGGGTCGCGAAAGGCCTTGTGGTACTG-3', in high stringency
hybridizations, according to the manual (Boehringer Mannheim GmbH,
Mannheim, Germany) and by genotyping. The genotypes were
determined either using the HCV LiPa protocol (Line Probe Assay
[LiPa]; Innogenetics, Ghent, Belgium),34 according to the
manual, or by direct-sequencing the products obtained by nested PCR
processing one tenth of the initial RT-PCR products. Nested PCR
(encompassing the same region as used in the HCV LiPa protocol) was
performed under the same conditions as the single PCR, but for 25 cycles, using sense primer (nt 74-91)
5'-AGCGCCTAGCCATGGCGT-3' and antisense primer (nt 243-260)
5'-TACCACAAGGCCTTTCGC-3', which were extended at the
5'-end with the 21 M13 primer and the reverse M13 primer,
respectively. The Thermo Sequenase cycle-sequencing reaction was
performed according to the manual, using the Dye-primer method
(Amersham Life Science, Buckinghamshire, UK). Serum samples were
initially used to determine the presence of HCV RNA before seroconversion. The finding of HCV RNA-positive samples before HCV
seroconversion were confirmed by detecting and genotyping HCV in
samples of serum and PBMCs or plasma drawn at the same time point.
Computer sequence analysis.
The PCR products were directly sequenced with an ABI Automated
Sequencer model 373A (Applied Biosystems, Columbia, MD) using the 1.2.0 software. The direct sequences were assembled by the Sequence Navigator program (ABI) and were further optimized
manually. P-distances and consensus sequences of part of the
5'UTR (based on at least 10 sequences of the 6 major genotypes
as found in Genbank) were calculated with MEGA.35
Phylogenetic trees were computed using the neighbour-joining
algorithm.36 The resulting tree was assessed by the
bootstrap method37 based on 500 replicates.
| |
RESULTS |
Determination of HCV seroconverters.
Testing of the first and the last samples drawn from 358 IDUs showed
that 316 (88.3%) were positive for antibodies to HCV, 23 (6.4%) were
negative for antibodies to HCV, and 19 (5.3%) seroconverted for HCV
during the follow-up period. One of 19 HCV seroconverters seroreverted
during follow-up, as described earlier.30 Antibodies to HCV
disappeared completely after the first 55 months, although HCV RNA was
intermittently detectable in serum during that period of 45 months.
Subsequently, a new seroconversion occurred at 98 months
(Fig 1).
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| Fig 1.
Patterns of HCV viremia and serological response in an
individual showing reseroconversion. The detection limit of the bDNA
assay is shown as a dotted line.
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Confirmation by hybridization of the initial HCV RNA-positive serum
samples in 19 HCV seroconverters.
To confirm all PCR products initially detected in serum samples of the
19 HCV seroconverters after UV illumination, we performed high-stringency Southern blot hybridizations using a
digoxigenine-labeled probe that disclosed the specificity of the
RT-PCR. The RT-PCR rendered good duplicates unless the detection limit
of the system was reached (Fig 2). As a
control panel, we included the first serum samples of 6 HCV
seronegative subjects who were seronegative with EIA 3.0 at both first
and last sample over a mean period of 5 years. RT-PCR and Southern blot
hybridization experiments performed on this control panel showed that
no detectable HCV RNA was present in any of the first samples (results
not shown).
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| Fig 2.
Southern blot hybridization of the duplicate RT-PCR
results using an internal 5'-digoxigenine-labeled HCV probe. The
initially detectable PCR products in duplicate of the 19 HCV
seroconverters were blotted and hybridized under high-stringency
conditions. The 19 seroconverters are indicated by an identification
number. Positive and negative controls are indicated.
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Presence of HCV RNA in different blood compartments among
HIV-negative subjects with prolonged antibody-negative periods.
RT-PCR was used to analyze serial blood samples for the presence of HCV
RNA before HCV seroconversion in different blood compartments drawn
from the 5 HIV-negative subjects (0073, 0146, 1083, 1085, and 3059),
when available. In 3 of these 5 individuals (0073, 1083, and 3059), the
initial sample was HCV RNA-negative. HCV RNA, as detected by single
RT-PCR, fluctuated but was repeatedly found at low levels in serial
samplings of all 5 subjects before HCV seroconversion. On some
occasions, HCV RNA was found by the bDNA assay in serum more than 1 year before HCV seroconversion. In 3 of 5 subjects (1085, 3059, and
0146), the presence of HCV RNA in serum could be confirmed
independently in PBMCs or plasma. The antibody status and presence of
HCV RNA in these 5 subjects are summarized in
Table 1.
Determination of HCV RNA by sequencing among subjects with prolonged
antibody-negative periods in different blood compartments.
To verify all positive findings by RT-PCR in the different blood
compartments from the 5 subjects, we performed sequence analyses of
samples drawn before and after HCV seroconversion. HCV genotypes were
determined using phylogenetic trees computed by the neighbour-joining algorithm, and genotype 1 was initially found in all subjects, although
sequences were distinct at 1 or more nucleotide positions. As shown in
Table 1, HCV RNA positivity was confirmed in independently collected
plasma or PBMC samples of 3 subjects (1085, 3059, and 0146), but
exclusively serum samples were available for testing in 2 subjects
(0073 and 1083).
In subject 1085, there was an extremely long antibody-undetectable
period of 94 months accompanied by intermittent detection of HCV RNA.
The initial serum sample, drawn 94 months before HCV seroconversion,
was positive for HCV RNA both by RT-PCR and bDNA testing and harbored
an HCV genotype 1. The sample collected 51 months before HCV
seroconversion was positive for HCV RNA by RT-PCR in serum and PBMCs
and contained HCV genotype 1 in both compartments. The sample taken at
47 months before seroconversion was negative for HCV RNA, whereas the
samples taken 4 months later were HCV RNA positive, and although HCV
genotype 1 was found in serum, HCV genotype 4 was found in PBMCs
sampled at the same time point. Thereafter, the HCV infection seemed to
be cleared from blood for the next 37 months. Infection with HCV
genotype 2 apparently occurred 6 months before HCV seroconversion, as
detectable in plasma and serum. The sample collected 2 months before
HCV seroconversion was HCV RNA negative, both in plasma and serum by
RT-PCR, whereas HCV genotype 1 was found just after HCV seroconversion,
indicating several reinfections in this individual
(Figs 3A and 4).
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| Fig 3.
Phylogenetic trees of the 5' noncoding region of
HCV showing the genotypes identified in 3 individuals with prolonged
antibody-undetectable periods. Patient specimens, consensus sequences,
time in months before () and after (+) HCV seroconversion, and
the 6 major consensus sequences are indicated. The trees were
bootstrapped 500 times and the numbers represent the percentages of the
500 bootstraps where those branches were the same. The horizontal
distances are represented by the scale bar in the lower part of the
figure and the distance is indicated.
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| Fig 4.
Alignment of all sequences found among the 3 individuals
with prolonged seronegative periods. Patient specimens, consensus
sequences, and time in months before () and after (+) HCV
seroconversion are indicated.
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In subject 3059, the antibody-undetectable period of 38 months was
accompanied by intermittent detection of HCV RNA. The sample taken at
38 months before HCV seroconversion was found to be positive by bDNA
and RT-PCR in serum and PBMCs. Sequencing of virus isolated from serum
and PBMC samples showed an infection with HCV genotype 1. HCV RNA
remained detectable in both compartments by RT-PCR and bDNA in the
sample taken at 34 months before HCV seroconversion; in PBMCs, genotype
1 was again detected by sequencing, but the virus in the serum sample
was untypeable by sequencing and InnoLipa. The serum sample taken at 31 months before HCV seroconversion was repeatedly negative by RT-PCR,
whereas the bDNA assay showed repeatedly positive results. HCV RNA was
not detectable by RT-PCR or bDNA in the next 3 serum samples taken over
a period of 17 months. Interestingly, in the sample drawn at 14 months
before HCV seroconversion, genotyping showed HCV genotype 1 in PBMCs, whereas in serum of the same time point, HCV genotype 3a was found. Moreover, in samples taken 2 months before HCV seroconversion, genotype
3a was found in serum, plasma, and PBMCs. HCV genotype 3a remained
present in the plasma samples tested after HCV seroconversion (Figs 3B
and 4).
In subject 0146, the antibody-undetectable period of 28 months was
accompanied by intermittent detection of HCV RNA. HCV genotype 1 was
found in serum samples 28 and 15 months before HCV seroconversion. The
serum sample collected 15 months before HCV seroconversion was positive
by RT-PCR and bDNA. Interestingly, 9 months before HCV seroconversion,
PBMCs were found to harbor HCV genotype 1, whereas the serum sample
appeared to be HCV RNA negative. The sample taken 2 months before HCV
seroconversion was HCV RNA-positive in serum and PBMCs, both of which
were infected with HCV genotype 4. However, serum samples up to 9 months after HCV seroconversion appeared to harbor HCV genotype 3a,
indicating several reinfections in this individual (Figs 3C and 4).
From subjects 0073 and 1083, only serum samples were available, and the
antibody-undetectable period was accompanied by intermittent detection
of HCV RNA. In subject 0073, HCV genotype 1 was detected in the serum
samples drawn 31 and 25 months before HCV seroconversion. The next 2 samples were HCV RNA negative, but HCV genotype 1 was again found in
consecutive serum samples drawn up to 25 months after HCV
seroconversion. In subject 1083, the antibody-undetectable period of 13 months was accompanied by intermittent detection of HCV RNA. The
initial serum sample, drawn 13 months before HCV seroconversion,
contained HCV genotype 1, whereas samples drawn at 8 and 6 months
before HCV seroconversion were HCV RNA-negative. Three months before
HCV seroconversion, the serum sample was positive for both RT-PCR and
bDNA, containing HCV genotype 1, and HCV seroconversion was observed in
the next sample, accompanied by undetectable HCV RNA. However, HCV
genotype 1 reappeared and was detectable both by RT-PCR and bDNA in the
consecutive plasma and serum samples.
| |
DISCUSSION |
Detection of antibodies directed to HCV is still considered the gold
standard for identification of HCV-infected individuals. A good
correlation has been shown between antibodies to HCV and detectable HCV
RNA, as well as between HCV seronegativity and undetectable RNA, among
blood donors,13,14 hemodialysis patients,16 hemophiliacs,17 and chronic HCV patients.19
However, up to half of immunosuppressed patients, such as recipients of
renal and liver transplants, fail to generate a humoral response if infected with HCV.25 Moreover, in studies of dialysis
patients38 and on histologically verified patients with
non-A, non-B hepatitis,19 commercially available antibody
tests failed to identify a number of HCV-infected individuals. Thomas
et al2 studied the sociodemographic and behavioral
correlates of HCV infections among IDUs in Baltimore and found a high
prevalence of HCV. They also found HCV RNA in 13 (30.2%) of 43 HCV-seronegative long-term drug users.
In the present study, we looked at the antibody-undetectable period for
HCV RNA to gain insight into the proportion of undiscovered HCV
carriers among IDUs. Because this population is at high risk for
coinfections with other parenterally transmittable agents, ie, HIV,
AIDS-associated immune-suppression could delay antibody response to HCV
infection. However, we found a long HCV-seronegative period
particularly in HIV-negative seroconverters (26.3%). The finding of
long antibody-undetectable periods before HCV seroconversion may be
explained in part by the transiently low detectable levels of HCV RNA
present in most IDUs before seroconversion, which are apparently in
some cases insufficient to elicit enough antibodies to be detected by
diagnostic assays. Nevertheless, our 5 subjects with prolonged
antibody-undetectable periods eventually seroconverted, apparently
regardless of HCV genotype and viral load. Whether our data can be
extrapolated to rarely exposed populations such as blood donors remains
unanswered. However, in some antibody-negative samples, we found HCV
RNA levels of more than 2 × 105 copies/mL that are in
the range of 105 to 109 copies/mL as usually
found in rejected blood donors during their preseroconversion periods
of approximately 85 days.39 Although some of our IDUs may
have comparable HCV RNA levels, results of quantification data obtained
from different study groups by different assays may be hard to
interpret. The question remains as to whether our observations warrant
screening of blood donors or suspected HCV-positive patients only for
antibodies to HCV by third-generation assays alone or in combination
with HCV RNA. Previous studies of us showed that some patients have
antibodies to HCV but low or no detectable HCV RNA in
blood.12 Moreover, full and partial HCV seroreversions,
with and without detectable HCV RNA, have been described in untreated
immunocompetent humans.29,30,40-42 Such seroreversions may
partially explain the antibody-undetectable period and may possibly
indicate resolved HCV infections from blood. Loss of antibodies to HCV
or intermittent seropositivity during follow-up may be interpreted as
resolution of HCV from blood or latent infection with low HCV RNA
levels and may be more common than generally suspected. Taken together,
screening for HCV by PCR must be used in the acute preseroconversion
phase, whereas screening for antibodies to HCV in combination with PCR may resolve all HCV-positive individuals. Thus, screening for antibodies to HCV in combination with PCR appears to be the safest way
to identify all HCV-infected individuals.
Improper handling and storage of specimens may affect the detection of
HCV RNA43,44 by yielding false-negative results, but such
artefacts would not shorten the antibody-undetectable periods in our
study. False RT-PCR-positive results can be introduced, either by
improper handling patient specimen or by improper extraction and
amplification of target RNA. To avoid such artefacts, the serum samples
on one hand and plasma and PBMC samples on the other hand were sent to
two different research institutes for final handling and storage.
Moreover, the serum and EDTA-blood samples of the 19 HCV seroconverters
were drawn at different Municipal Health stations in Amsterdam. To
exclude false-positive serum results due to sampling, handling, or
storage, plasma and PBMC samples were also tested when available.
Moreover, all samples were randomly tested for the presence of HCV RNA,
and all precautions were taken to avoid any possible contamination,
using separated locations for the extraction of specimens,
amplification of HCV RNA (using the GeneAmp PCR carry-over prevention
kit), and analyses of PCR products.
In general, serum and PBMC samples drawn and analyzed at the same
time-point contained the same HCV genotype. However, at some time
points in subjects 1085 and 3059, the serum sample contained another
HCV genotype than the genotype found in PBMCs. Especially remarkable
findings were observed in samples taken before HCV seroconversion in
subject 3059. In that patient, HCV genotype 1 was initially found in
serum and PBMCs 38 months before HCV seroconversion. HCV genotype 1 remained detectable in the 2 following PBMC samples, whereas the serum
samples were either untypeable or HCV-negative by RT-PCR. However, 14 months before HCV seroconversion, genotype 1 remained present in PBMCs,
whereas the serum sample of that time contained genotype 3a.
Strikingly, the samples drawn 2 months before HCV seroconversion
contained genotype 3a in serum, plasma, and PBMCs, indicating a new
infection that was found in all consecutive samples after HCV
seroconversion. These findings could be explained by the fact that
frequent drug injections lead to a mixture of HCV genotypes. Serotypes
of developing antibodies may show how to interpret the final
seroconversions observed. This is demonstrated by an earlier study by
us in which a comparison was made between the obtained HCV genotypes
and serotypes of the antibodies as they developed based on the antibody
response to core and NS4 in the 19 HCV seroconverters.45
The serotypes we found were in concordance with the HCV genotypes found
after HCV seroconversion IDU 0146 (genotype 3a), 1085 (genotype 1), and 3059 (genotype 3a) and not with HCV genotypes found at several samples
drawn during the prolonged antibody-undetectable periods. The serotypes
did not change during follow-up, regardless of infections with
heterologous HCV genotypes.45 Continuous reexposure and reinfections with HCV are often seen among IDUs, as exemplified in
individual 3009 (Fig 1), in whom a new HCV seroconversion was detected
by commercially available antibody assays. Superinfections and overtake phenomena in seropositive subjects have been reported in
chimpanzees experimentally reinfected with HCV,24,46,47 in
chronically HCV-positive humans reinfected with HCV through blood
transfusion,48 and in chronically HCV-positive humans receiving HCV-positive liver grafts.49 According to these
reports, neither humans nor chimpanzees chronically infected with HCV
have adequate protective immunity against heterologous HCV genotypes or
are protected against HCV strains of the same genotype or even against
identical strains.
Another explanation, in addition to low levels of HCV RNA, might be a
poor recognition or exposure of HCV antigens to helper T lymphocytes
(TH cells), leading to diminished lymphokine secretion and
impaired expansion, growth, and responsiveness of B cells. Among the 5 HIV-negative individuals, no differences were found in T-cell numbers
during periods of at least 1 year before and after HCV seroconversion
(results not shown). However, it has been described that exposure to
opiates negatively influences T-cell responses, especially anti-CD3
responses. IDUs have reduced reactivity of T cells and higher levels of
anergy than non-IDUs.50 This might be particularly relevant
to our study, because long antibody-undetectable periods in our study
were seen among HIV-negative individuals, excluding HIV as confounding
factor. Extended antibody-undetectable periods were also found for HIV
among IDUs compared with non-IDUs infected with HIV, although the delay
between HIV detection and seroconversion is not as extreme as with
HCV.51
In conclusion, we observed a prolonged period of HCV RNA-positivity
before antibody seroconversion in a number of injecting drug users.
Independent of HIV infection, the immune system appears to be sometimes
incompetent to mount enough antibodies to HCV to be detected by
commercially available assays, making antibody screening alone prone to
false-negative results in the diagnosis of HCV infection. Our results
indicate that HCV infection is not always characterized by persistent
antibody responses and can be sustained at low levels without or
delayed antibody responses to HCV. Therefore, we suggest screening for
antibodies to HCV in combination with PCR to identify all HCV-positive
individuals and potentially infectious blood donations. However, the
percentage of silent carriers is hard to predict, and more studies are
needed in subjects other than IDUs to extrapolate these findings to the general population.
| |
ACKNOWLEDGMENT |
The authors thank Lucy Phillips for editorial review, Martin McMorrow
(Chiron Diagnostics) for bDNA assays, and Wim van Est for fine artwork.
| |
FOOTNOTES |
Submitted December 2, 1998; accepted April 5, 1999.
Supported by the Health Research and Development Council (28-2370) and
performed as part of the Amsterdam Cohort Studies on AIDS, a
collaboration between the Academic Medical Centre, the Central
Laboratory of the Netherlands Red Cross Blood Transfusion Service, and
the Municipal Health Service, Amsterdam, The Netherlands. Approval was
obtained from the Institutional Review Board for these studies.
Informed consent was provided according to the Declaration of Helsinki.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jaap Goudsmit, MD, PhD,
Department of Human Retrovirology, Academic Medical Centre, University
of Amsterdam Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands;
e-mail: J.Goudsmit{at}AMC.UvA.NL.
| |
REFERENCES |
1.
Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M:
Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.
Science
244:359, 1989[Abstract/Free Full Text]
2.
Thomas DL, Vlahov D, Solomon L, Cohn S, Taylor E, Garfein R, Nelson KE:
Correlates of hepatitis C virus infections among injecting drug users.
Medicine
74:212, 1995[Medline]
[Order article via Infotrieve]
3.
van den Hoek JAR, Goudsmit J, de Wolf F, Coutinho RA:
Prevalence, incidence and risk factors of hepatitis C virus infection among drug users in Amsterdam.
J Infect Dis
162:823, 1990[Medline]
[Order article via Infotrieve]
4.
Rezza G, Sagliocca L, Zaccarelli M, Nespoli M, Siconolfi M, Baldassarre C:
Incidence rate and risk factors for HCV seroconversion among injecting drug users in an area with low HIV seroprevalence.
Scan J Infect Dis
28:27, 1996[Medline]
[Order article via Infotrieve]
5.
Brunetto MR, Calvo PL, Oliveri F, Colombatto P, Abate ML, Manzini P, Bonino F:
Hepatitis C virus infection and liver disease: Peculiar epidemiological and clinicopathological features.
FEMS Microbiol Rev
14:259, 1994[Medline]
[Order article via Infotrieve]
6.
Esteban R:
Epidemiology of hepatitis C virus infection.
J Hepatol
17:S67, 1993 (suppl 3)
7.
De Mitri MS, Poussin K, Baccarini P, Pontisso P, D'Errico A, Simon N, Grigioni W, Alberti A, Beaugrand M, Pisi E, Brechot C, Paterlini P:
HCV-associated liver cancer without cirrhosis.
Lancet
345:413, 1995[Medline]
[Order article via Infotrieve]
8.
Saito I, Miyamura T, Ohbayashi A, Harada H, Katayama T, Kikuchi S, Watanabe Y, Koi S, Onji M, Ohta Y, Choo QL, Hughton M, Kuo G:
Hepatitis C virus infection is associated with the development of hepatocellular carcinoma.
Proc Natl Acad Sci USA
87:6547, 1990[Abstract/Free Full Text]
9.
Alter HJ, Sanchez-Pescador R, Urdea MS, Wilber JC, Lagier RJ, Di Bisceglie AM, Shih JW, Neuwald PD:
Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA.
J Vir Hepatitis
2:121, 1995[Medline]
[Order article via Infotrieve]
10.
Young KK, Resnick RM, Myers TW:
Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay.
J Clin Microbiol
31:882, 1993[Abstract/Free Full Text]
11.
Mitsui T, Iwano K, Masuko K, Yamazaki C, Okamoto H, Tsuda F, Tanaka T, Mishiro S:
Hepatitis C virus infection in medical personnel after needlestick accident.
Hepatology
16:1109, 1992[Medline]
[Order article via Infotrieve]
12.
Beld M, Penning M, McMorrow M, Gorgels J, van den Hoek A, Goudsmit J:
Different hepatitis C virus (HCV) RNA load profiles following seroconversion among injecting drug users without correlation with HCV genotype and serum alanine aminotransferase levels.
J Clin Microbiol
36:872, 1998[Abstract/Free Full Text]
13.
Lavanchy D, Mayerat C, Morel B, Schneider P, Zufferey C, Gonvers JJ, Pecoud A, Frei PC:
Evaluation of third-generation assays for detection of anti-hepatitis C virus (HCV) antibodies and comparison with presence of HCV RNA in blood donors reactive to c100-3 antigen.
J Clin Microbiol
32:2272, 1994[Abstract/Free Full Text]
14.
Zaaijer HL, Vrielink H, van Exel-Oehlers PJ, Cuypers HT, Lelie PN:
Confirmation of hepatitis C infection: A comparison of five immunoblot assays.
Transfusion
34:603, 1994[Medline]
[Order article via Infotrieve]
15.
Conry-Cantalena C, VanRaden M, Gibble J, Melpolder J, Obaid Shakil A, Viladomiu L, Ling C, Di Bisceglie A, Hoofnagle J, Kaslow R, Ness P, Alter HJ:
Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection.
N Engl J Med
334:1691, 1996[Abstract/Free Full Text]
16.
Courouce 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-R NA and anti-HCV antibodies (third-generation assays).
Nephrol Dial Transpl
10:234, 1995[Abstract/Free Full Text]
17.
Mauser-Bunschoten EP, Bresters D, van Drimmelen AA, Roosendaal G, Cuypers HT, Reesink HW, van der Poel CL, van den Berg HM, Lelie PN:
Hepatitis C infection and viremia in Dutch hemophilia patients.
J Med Virol
45:241, 1995[Medline]
[Order article via Infotrieve]
18.
Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, Rosenblatt J, Choo QL, Houghton M:
Detection of hepatitis C viral sequences in non-A, non-B hepatitis.
Lancet
335:1, 1990[Medline]
[Order article via Infotrieve]
19.
Kao JH, Lai MY, Hwang YT, Yang PM, Chen PJ, Sheu JC, Wang TH, Hsu HC, Chen DS:
Chronic hepatitis C without anti-hepatitis C antibodies by second-generation assay. A clinicopathologic study and demonstration of the usefulness of a third-generation assay.
Dig Dis Sci
41:161, 1996[Medline]
[Order article via Infotrieve]
20.
Lelie PN, Cuypers HT, Reesink HW, van der Poel CL, Winkel I, Bakker E, van Exel-Oehlers PJ, Vallari D, Allain JP, Mimms L:
Patterns of serological markers in transfusion-transmitted hepatitis C virus infection using second-generation HCV assays.
J Med Virol
37:203, 1992[Medline]
[Order article via Infotrieve]
21.
Barrera JM, Bruguera M, Ercilla MG, Gil C, Celis R, Gil MP, del Valle Onorato M, Rodes J, Ordinas A:
Persistent hepatitis C viremia after acute self-limiting posttransfusion hepatitis C.
Hepatology
21:639, 1995[Medline]
[Order article via Infotrieve]
22.
Esteban JI, Gomez J, Martell M, Cabot B, Quer J, Camps J, Gonzalez A, Otero T, Moya A, Esteban R, Guardia J:
Transmission of hepatitis C virus by a cardiac surgeon.
N Engl J Med
334:555, 1996[Abstract/Free Full Text]
23.
van Doorn LJ, Quint W, Tsiquaye K, Voermans J, Paelinck D, Kos T, Maertens G, Schellekens H, Murray K:
Longitudinal analysis of hepatitis C virus infection and genetic drift of the hypervariable region.
J Infect Dis
169:1226, 1994[Medline]
[Order article via Infotrieve]
24.
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
258:135, 1992[Abstract/Free Full Text]
25.
Maple PA, McKee T, Desselberger U, Wreghitt TG:
Hepatitis C virus infections in transplant patients: serological and virological investigations.
J Med Virol
44:43, 1994[Medline]
[Order article via Infotrieve]
26.
Schmidt WN, Wu P, Cederna J, Mitros FA, LaBrecque DR, Stapleton JT:
Surreptit ous hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative antibody tests.
J Infect Dis
176:27, 1997[Medline]
[Order article via Infotrieve]
27.
Schmidt WN, Wu P, Han J-Q, Perino MJ, LaBrecque DR, Stapleton JT:
Distribution of hepatitis C virus (HCV) RNA in whole blood and blood cell fractions: Plasma HCV RNA analysis underestimates circulating virus load.
J Infect Dis
176:20, 1997[Medline]
[Order article via Infotrieve]
28.
Bassett SE, Brasky KM, Lanford RE:
Analysis of hepatis C virus-inoculated chimpanzees reveals unexpected clinical profiles.
J Virol
72:2589, 1998[Abstract/Free Full Text]
29.
Lefrère J-J, Guiramand S, Lefrere F, Mariotti M, Aumont P, Lerable J, Petit J, Girot R, Morand-Joubert L:
Full or partial seroreversion in patients infected by hepatitis C virus.
J Infect Dis
175:316, 1997[Medline]
[Order article via Infotrieve]
30.
Beld M, Penning M, van Putten M, Lukashov V, van den Hoek A, McMorrow M, Goudsmit J:
Quantitative antibody responses to structural (core) and nonstructural (NS3, NS4, and NS5) hepatitis C virus proteins among seroconverting injecting drug users: Impact of epitope variation and relationship to detection of HCV RNA in blood.
Hepatology
29:1288, 1999[Medline]
[Order article via Infotrieve]
31.
van den Hoek JAR, Coutinho RA, van Haastrecht HJ, van Zadelhoff AW, Goudsmit J:
Prevalence and risk factors of HIV infections among drug users and drug-using prostitutes in Amsterdam.
AIDS
2:55, 1988[Medline]
[Order article via Infotrieve]
32.
Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J:
Rapid and simple method for purification of nucleic acids.
J Clin Microbiol
28:495, 1990[Abstract/Free Full Text]
33.
Attia MAM, Zekri A, Goudsmit J, Boom R, Khaled HM, Mansour MT, de Wolf F, El-Din HM, Sol CJA:
Diverse patterns of recognition of hepatitis C virus core and non-structural antigens by antibodies present in Egyptian cancer patients and blood donors.
J Clin Microbiol
34:2665, 1996[Abstract]
34.
Stuyver L, Rossau R, Wyseur A, Duhamel M, Vanderborght B, Van Heuverswyn H, Maertens G:
Typing of hepatitis C virus isolates and characterization of new subtypes using a line probe assay.
J Gen Virol
74:1093, 1993[Abstract/Free Full Text]
35.
Kumar S, Tamura K, Nei M:
MEGA: Molecular evolutionary genetics analysis software for microcomputers.
Computer Appl Biosci
10:189, 1994[Abstract/Free Full Text]
36.
Saitou N, Nei M:
The neighbor-joining method: A new method for reconstructing phylogenetic trees.
Mol Biol Evol
4:406, 1987[Abstract]
37.
Felsenstein J:
Phylogenies from molecular sequences: Inference and reliability.
Annu Rev Genet
22:521, 1988[Medline]
[Order article via Infotrieve]
38.
Bukh J, Wantzin P, Krogsgaard K, Knudsen F, Purcell RH, Miller RH:
High prevalence of hepatitis C virus (HCV) RNA in dialysis patients: Failure of commercially available antibody tests to identify a significant number of patients with HCV infection. Copenhagen Dialysis HCV Study Group.
J Infect Dis
168:1343, 1993[Medline]
[Order article via Infotrieve]
39.
Nübling CM, Seitz R, Löwer J:
Application of nucleic acid amplification techniques for blood donation screening.
Infusion Ther Transfusion Med
25:86, 1998
40.
Tan D, Im SW, Peng WW, Ng MH:
Follow-up study of acute hepatitis C.
Arch Virol
138:71, 1994[Medline]
[Order article via Infotrieve]
41.
Lai ME, Mazzoleni AP, Argiolu F, De Virgilis S, Balestrieri A, Purcell RH, Cao A, Farci P:
Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children.
Lancet
343:388, 1994[Medline]
[Order article via Infotrieve]
42.
Aquilar C, Lucia JF:
Anti-HCV seroreversion in HIV-negative haemophiliacs.
Br J Haematol
93:497, 1996
43.
Busch MP, Wilber JC, Johnson P, Tobler L, Evans CS:
Impact of specimen handling and storage on detection of hepatitis C virus RNA.
Transfusion
32:420, 1992[Medline]
[Order article via Infotrieve]
44.
Cuypers HT, Bresters D, Winkel IN, Reesink HW, Weiner AJ, Houghton M, van der Poel CL, Lelie PN:
Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.
J Clin Microbiol
30:3220, 1992[Abstract/Free Full Text]
45.
Beld M, Penning M, van Putten M, van den Hoek A, Lukashov V, McMorrow M, Goudsmit J:
Hepatitis C virus serotype-specific Core and NS4 antibodies in injecting drug users participating in the Amsterdam cohort studies.
J Clin Microbiol
36:3002, 1998[Abstract/Free Full Text]
46.
Okamoto H, Mishiro S, Tokita H, Tsuda F, Miyakawa Y, Mayumi M:
Superinfection of chimpanzees carrying hepatitis C virus of genotype II/1b with that of genotype III/2a or I/1a.
Hepatology
20:1131, 1994[Medline]
[Order article via Infotrieve]
47.
Prince AM, Brotman B, Huima T, Pascual D, Jaffery M, Inshauspé G:
Immunity in hepatitis C infection.
J Infect Dis
165:438, 1992[Medline]
[Order article via Infotrieve]
48.
Kao JH, Chen PJ, Lai MY, Chen DS:
Superinfection of heterologous hepatitis C virus in a patient with chronic type C hepatitis.
Gastroenterology
105:583, 1993[Medline]
[Order article via Infotrieve]
49.
Laskus T, Wang LF, Rakela J, Vargas H, Pinna AD, Tsamandas AC, Demetris AJ, Fung J:
Dynamic behavior of hepatitis C virus in chronically infected patients receiving liver graft from infected donors.
Virology
220:171, 1996[Medline]
[Order article via Infotrieve]
50.
Mientjes GH, Miedema F, van Ameijden EJ, van den Hoek JAR, Schellekens PTA, Roos MT, Coutinho RA:
Frequent injecting impairs lymphocyte reactivity in HIV-positive and HIV-negative drug users.
AIDS
5:35, 1991[Medline]
[Order article via Infotrieve]
51.
Bruisten SM, Mientjes GH, van Delft P, de Wolf F, van Ameijden EJ, Cornelissen M, Goudsmit J, Coutinho RA, Huisman JG:
HIV-1 infection detected by polymerase chain reaction frequently precedes antibody seroconversion in drug users (letter).
AIDS
8:1736, 1994[Medline]
[Order article via Infotrieve]
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