HCV is Detected in Liver of HCV Antibody-Positive But HCV RNA Negative Patients,

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HCV is Detected in Liver of HCV Antibody-Positive But HCV RNA

Negative Patients, so don't assume HCV has been cleared if viral load

is undetectable

“Detection of Hepatitis C Virus (HCV) RNA in the Liver of Healthy,

Anti-HCV Antibody-Positive, Serum HCV RNA-Negative Patients with

Normal Alanine Aminotransferase Levelsâ€

“…..the majority of healthy patients who test positive for anti-

HCV antibodies and have normal ALT levels but who do not have HCV RNA

detected in serum have an ongoing HCV infection because HCV RNA is

detected in the liver. The epidemiological and clinical relevance of

this finding should be studied in the future…..â€

The Journal of Infectious Diseases July 1, 2006;194:53-60

Vicente Carreño, Margarita Pardo, López-Alcorocho,

Elena Rodríguez-Iñigo, Bartolomé, and Inmaculada Castillo

Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain

ABSTRACT

Background. It is unknown whether hepatitis C virus (HCV) is

present in the liver of anti-HCV antibody-positive patients with

persistently normal alanine aminotransferase (ALT) levels and

undetectable serum HCV RNA levels.

Methods. We determined the presence of genomic and antigenomic

HCV RNA strands in liver biopsy specimens and peripheral blood

mononuclear cell (PBMC) samples obtained from 12 anti-HCV antibody-

positive patients who had normal ALT levels and who had been serum

HCV RNA negative for at least 12 months, according to the results of

quantitative, strand-specific, real-time reverse-transcription-

polymerase chain reaction and, also, in situ hybridization of liver

cells. Intrahepatic HCV RNA was cloned and sequenced.

Results.

All patients remained anti-HCV antibody positive and serum HCV RNA

negative, and all had normal ALT values during follow-up (mean

duration ± SD, 29.2 ± 19.8 months).

Genomic HCV RNA was detected in liver biopsy specimens obtained from

10 (83%) of 12 patients, and the antigenomic strand was detected in

10 (100%) of 10 liver biopsy specimens in which genomic HCV RNA was

detected.

Results were confirmed by in situ hybridization. Intrahepatic HCV was

of genotype 1b, and HCV sequencing demonstrated no cross-

contamination among samples. Genomic HCV RNA was found in 6 (50%) of

12 PBMC samples, and antigenomic HCV RNA was also detected in 5 (83%)

of these 6 PBMC samples.

Conclusion. HCV may persist and replicate in the liver and PBMCs

of healthy, anti-HCV antibody-positive, serum HCV RNA-negative

patients who have persistently normal ALT levels. These patients

should be followed up, because they have an ongoing viral infection.

Hepatitis C virus (HCV) infection is a major cause of liver disease

that is characterized by the presence of antibodies against HCV

proteins (i.e., anti-HCV antibodies) and HCV RNA in serum [1]. During

acute HCV infection, 15%-20% of individuals clear the virus, with

loss of serum HCV RNA and normalization of liver-function test

results occurring but with anti-HCV antibodies remaining detectable

[2]. Clearance of HCV infection also occurs in individuals with

chronic hepatitis C, after receipt of successful antiviral treatment

[3]. All of these individuals are considered to have completely

recovered from HCV infection and to have been immunized against at

least the corresponding HCV strain [4]. On the other hand, there are

healthy individuals with normal alanine aminotransferase (ALT) levels

who test positive for anti-HCV antibodies in the absence of serum HCV

RNA and who have no history of acute or chronic liver disease [5].

This situation could reflect an immunological response to an

unapparent exposure to HCV without clinical consequences, rather than

a clearance of a HCV infection.

Recently, a new form of HCV infection, defined as " occult HCV

infection, " has been described [6]. Occult HCV infection is

characterized by the absence of anti-HCV antibodies and serum HCV RNA

and by the presence of HCV RNA in the peripheral blood mononuclear

cells (PBMCs) of most patients (70%) and in the liver of all

patients. In addition, detection of HCV RNA in the PBMCs of patients

with anti-HCV antibodies and normal ALT levels years after the

resolution of HCV infection (either spontaneously or induced by

antiviral treatment) has also been reported [7-9]. Also, the presence

of intrahepatic HCV RNA after normalization of liver enzyme levels

and clearance of serum HCV RNA in patients with chronic hepatitis C

who responded to an antiviral treatment has been demonstrated [9,

10]. However, up to now, the possible presence of HCV RNA in the

liver tissue of healthy, anti-HCV antibody-positive patients with

persistently normal ALT levels and no history of acute or chronic

liver disease has not been studied. In the present study, we analyzed

whether anti-HCV antibody-positive, serum HCV RNA-negative patients

with persistently normal ALT levels have HCV RNA (genomic HCV RNA) in

the liver and, also, whether HCV is replicating by detecting the

antigenomic HCV RNA.

DISCUSSION

Anti-HCV antibody-positive, serum HCV RNA-negative patients who have

normal ALT levels may be considered to be patients who have cleared

HCV infection, either spontaneously or after successful antiviral

treatment [2, 3]. However, recent studies [7-10] have demonstrated

that HCV RNA may be present in the liver or PBMCs of such patients,

thus indicating an ongoing HCV infection. To our knowledge, no

studies have been performed to search for the presence of HCV RNA in

the liver of healthy, anti-HCV antibody-positive, serum HCV RNA-

negative patients with normal ALT levels who do not have a previous

history of acute or chronic liver disease.

In the present study, HCV RNA was detected in the liver biopsy

specimens of 10 (83%) of 12 healthy, anti-HCV antibody-positive

patients with persistently normal ALT levels and without HCV RNA

detected in serum. The specificity of the results was demonstrated by

sequencing data that showed the absence of cross-contamination among

the samples. Furthermore, all results were confirmed by in situ

hybridization. It should be noted that 8 of the 10 patients with

intrahepatic HCV RNA did not have risk behaviors for HCV infection,

whereas the other 2 patients had received a blood transfusion >25

years previously.

As mentioned above, previous studies [9, 10] have also reported the

presence of HCV RNA in the liver of patients who cleared HCV

infection after receiving successful antiviral treatment, albeit in

lower percentages (2% and 27%) than were noted in our study. Other

than the differences in the populations studied, several factors

could explain this discrepancy. One factor could be possible HCV RNA

degradation resulting from improper preservation of liver specimens,

with this leading to false-negative results [15]. Although

McHutchison et al. [10] analyzed positive internal controls to ensure

the integrity of the RNA in order to validate their PCR results, it

has been proved that amplification of positive internal controls does

not assure the integrity of HCV RNA [15]. Another cause could be the

different methodology employed for HCV RNA isolation as well as for

HCV RNA amplification.

In addition, the antigenomic HCV RNA strand was detected in 10 of 10

liver samples that had genomic viral RNA. This result demonstrates

that HCV was replicating in liver cells in the majority of patients

(83%) who had no history of liver disease but who tested positive for

anti-HCV antibodies in the absence of HCV RNA in serum and who

presented with constantly normal ALT levels. Therefore, it should be

considered that, once HCV infects the liver, complete clearance of

HCV is not a frequent event, at least in untreated patients. In light

of these results, anti-HCV antibody-positive, serum HCV RNA-negative

patients with normal ALT levels who are considered to have cleared

HCV may, in fact, have an ongoing HCV infection. Thus, healthy

patients with normal ALT levels who are found to be anti-HCV antibody

positive but serum HCV RNA negative during routine screening for anti-

HCV antibodies (i.e., for blood donations, pregnancy, epidemiological

studies, or medical checkups) should be carefully followed up.

HCV RNA was also detected in 50% of the PBMC samples obtained from

our anti-HCV antibody-positive, serum HCV RNA-negative patients who

had a normal ALT level. The frequency of HCV infection noted in PBMC

samples in the present study agrees with that reported by Radkowski

et al. [8], who found HCV RNA in uncultured PBMC samples obtained

from 7 of 11 patients who also had normal ALT values and anti-HCV

antibodies but who did not have detectable viral RNA in serum,

although these authors did not study liver biopsy specimens. Also, 5

of our 6 patients with the genomic HCV RNA strand detected in PBMC

samples also had the antigenomic HCV RNA strand; therefore, viral

replication was taking place in these cells. In light of these

results, a proportion of anti-HCV antibody-positive patients who have

a normal ALT level and are serum HCV RNA negative should be

considered to be potentially infectious. An epidemiological study

should be performed to confirm this hypothesis. In addition, patients

who are anti-HCV antibody positive and have normal ALT levels but who

do not have HCV RNA detected in PBMC samples should also be

prospectively studied, because most of these patients have HCV

replication in the liver, and, thus, it could be that HCV virions may

be released to the bloodstream during cytotoxic or immunosuppressive

therapy. In support of this hypothesis, the case of a single patient

who cleared HCV infection and, after 8.5 years of quiescence with

negative serum HCV RNA results, had serum HCV RNA results again

become positive (with the same virus as originally observed) after

receipt of prednisone therapy has recently been published [16].

Regarding the liver histological findings for the 10 patients with

intrahepatic HCV RNA, 2 of the patients had nonalcoholic

steatohepatitis and 1 had steatosis, apparently not in association

with HCV infection, because these patients either were overweight or

had a metabolic disorder. Six other patients had minimal histological

changes. These results suggest that, despite the presence and

replication of HCV in the liver of these patients, the histological

lesion is minimal. However, 1 patient (10%) had chronic active

hepatitis with liver fibrosis diagnosed, and, thus, the natural

history and evolution of these patients should be studied.

In summary, the majority of healthy patients who test positive for

anti-HCV antibodies and have normal ALT levels but who do not have

HCV RNA detected in serum have an ongoing HCV infection because HCV

RNA is detected in the liver. The epidemiological and clinical

relevance of this finding should be studied in the future.

RESULTS

For the 12 anti-HCV antibody-positive patients with normal ALT levels

who were included in the present study, HCV RNA remained undetectable

(as tested by real-time RT-PCR) in the serum sample collected at the

same time that the liver biopsy specimen was obtained. The genomic

HCV RNA strand was detected in liver specimens obtained from 10 (83%)

of 12 patients. Regarding HCV replication, the antigenomic HCV RNA

strand was found in 10 (100%) of 10 patients who had genomic HCV RNA

detected in the liver biopsy specimen (table 3). No HCV RNA was

detected in any of the negative controls included in the assays, and

the results of HCV RNA detection performed by different operators on

different days were identical in all cases. The mean load of the

genomic HCV RNA strand was significantly higher (P = .005) than that

of the antigenomic strand (2.6 × 105 ± 1.9 × 105 vs. 1.1 × 105 ±

1.0 × 105 copies/ug total RNA, respectively). The presence of

genomic and antigenomic HCV strands was confirmed in all cases by in

situ hybridization (table 3). Finally, no correlation was found

between anti-HCV antibody titers and the amount of genomic or

antigenomic HCV RNA strands in the liver.

Genotyping of intrahepatic HCV RNA showed that the 10 patients with

viral RNA detected in liver biopsy specimens had HCV of genotype 1b.

Nucleotide sequence analysis of the HCV core region from 5 randomly

selected patients confirmed that HCV isolates belonged to genotype

1b. The phylogenetic analysis of the HCV core region demonstrated

that the genetic distances of the clones within patients were lower

than those among patients (table 4). The phylogenetic tree showed

that the 4 clones segregated separately in the 5 patients, indicating

that no cross-contamination among samples occurred (figure 2).

Regarding PBMCs, the genomic HCV RNA strand was detected in 6 (50%)

of 12 patients, whereas HCV replication (antigenomic HCV RNA strand)

was demonstrated in 5 (83%) of the 6 PBMC samples from the patients

who had the genomic HCV RNA strand detected in these cells (table 3).

Again, the mean load of the genomic HCV RNA strand was significantly

higher (P = .043) than that of the antigenomic HCV RNA strand (2.2 ×

105 ± 1.9 × 105 vs. 1.5 × 105 ± 8.5 × 104 copies/g total RNA,

respectively). The 2 patients who did not have HCV RNA in their liver

biopsy specimens also did not have HCV RNA in their PBMC samples.

With respect to histological findings for liver biopsy samples, 6

(60%) of the 10 patients who had HCV RNA detected had minimal

histological changes (table 3). Two other patients (20%) had

nonalcoholic steatohepatitis (one who had hypercholesterolemia and

was overweight and another who was overweight), and one patient had

steatosis (with hyperlipidemia). The remaining patient with HCV RNA

detected in a liver biopsy specimen presented with chronic active

hepatitis, with grade 2 portal activity, grade 2 lobular activity,

and stage 1 fibrosis. For this patient, other causes of liver damage

(hepatitis B virus infection, autoimmunity, drug toxicity, metabolic

and genetic disorders, and alcohol intake) were ruled out on the

basis of clinical and analytical data. The 2 patients who did not

have HCV RNA in liver biopsy specimens had minimal histological

changes.

PATIENTS AND METHODS

Of a total of 95 anti-HCV antibody-positive, serum HCV RNA-negative

individuals with normal ALT levels (for at least 12 months) who were

attending our center (Fundación para el Estudio de las Hepatitis

Virales, Madrid, Spain), 12 patients underwent a programmed

interventional laparoscopy (of the gallbladder [10 patients] and the

esophageal hiatal hernia [2 patients]). These 12 individuals were

included in the present study because they gave their written,

informed consent for a liver specimen to be obtained during the

laparoscopy. The study was conducted in accordance with the

principles of the Declaration of Helsinki. The demographic and

clinical characteristics of the 12 case patients, according to the

day when the liver biopsy specimen was obtained, are shown in table 1.

Table 1. Characteristics of the 12 healthy subjects who were anti-

hepatitis C virus (HCV) antibody positive and serum HCV RNA negative

on the day when the liver biopsy specimen was obtained.

Screening for anti-HCV antibodies in these patients was done for

several reasons (table 1), and, thereafter, the patients were

referred to our institution, where the presence of anti-HCV

antibodies was confirmed for all case patients by use of a commercial

recombinant immunoblot assay (Innolia HCV Ab III; Innogenetics). In

addition, all patients were serum HCV RNA negative, as was determined

by a commercial test (Amplicor HCV, version 2.0 [Roche Diagnostics];

test sensitivity, 50 IU/mL; test specificity, 99%), and all had

normal transaminase levels. None of these 12 patients had a clinical

history of acute or chronic liver disease, and, except for 2 patients

who had received a blood transfusion >25 years previously, they did

not report risk factors for HCV infection. This first visit to our

institution was considered to be the first day of the follow-up,

which had a mean duration (±SD) of 29.2 ± 19.8 months. During the

whole follow-up, the 12 anti-HCV antibody-positive patients continued

to have persistently normal ALT levels and were serum HCV RNA

negative, as was determined every 6 months. As was previously

mentioned, a liver biopsy specimen was obtained from all these

patients at a mean time (±SD) of 22.1 ± 13.6 months after the

beginning of the follow-up. Table 2 presents data on transaminase

levels at 3 time points during follow-up, for each of the 12 patients.

After the liver biopsy fragment was obtained, it was cut into 2

portions. The first portion was used for histological diagnosis [11],

and the second portion was submerged (no later than 30 s after the

liver specimen was obtained) into RNAlater (Ambion) and then was

stored at -20°C until it was used for HCV RNA detection.

Serum and PBMC samples were collected from all patients on the same

day when laparoscopy was performed. Transaminase levels were tested

again, and anti-HCV antibody titers were determined by means of

serial dilutions (1 : 100 to 1 : 100,000) of the serum samples, by

use of a commercial kit (Innotest-HCV Ab IV; Innogenetics). Finally,

an aliquot of serum was stored at -80°C, whereas PBMCs were stored

in RNAlater (Ambion) at -20°C until they were used for HCV RNA

detection.

Detection of genomic and antigenomic HCV RNA strands by quantitative,

strand-specific, real-time reverse-transcription-polymerase chain

reaction (RT-PCR). Total RNA was isolated from 200 L of serum,

from liver specimens, and from PBMC samples, by use of the SV Total

RNA Isolation kit (Promega). After precipitation, the pellet was

dissolved in diethyl pyrocarbonate-treated distilled water. The total

RNA concentration from the liver and PBMC samples was determined

using spectrophotometry.

Quantification of the 5 noncoding region of the genomic and

antigenomic HCV RNA strands was done using a strand-specific real-

time RT-PCR, with use of the thermostable enzyme Tth for the

synthesis of cDNA at a high temperature. Thus, for the amplification

of the genomic HCV RNA strand, cDNA was generated in 20 L of reaction

mixture that contained the total RNA extracted from 200 uL of serum

or 0.5 ug of total RNA from liver specimens or PBMC samples, 50

pmol/L antisense primer UTRLC2 (5'-CAAGCACCCTATCAGGCAGT-3'), 1 mmol/L

MnCl2, 200 umol/L each deoxynucleotide triphosphate, 1× RT buffer

(Applied Biosystems), and 5 U of Tth (Applied Biosystems). After 20

min at 65°C, the RT activity was inactivated by Mn2+ chelation with

8 uL of the 10× chelating buffer (Applied Biosystems), followed by

heating at 95°C for 30 min. For amplification of antigenomic HCV

RNA, cDNA was synthesized under the same conditions by the addition

of 50 pmol/L sense primer UTRLC1 (5'-CTTCACGCAGAAAGCGTCTA-3'). Real-

time PCR was run in a LightCycler (Roche Molecular Biochemicals) with

2 L of cDNA in a final volume of 20 uL, with use of the LightCycler

FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals).

The reaction mixture contained 4 mmol/L MgCl2, 0.5 umol/L primers

UTRLC1 and UTRLC2, and 2 uL of SYBR Green Master mix. Amplification

was performed as follows: initial denaturation and activation of

enzyme were performed at 95°C for 10 min; followed by 60 cycles at

95°C for 1 s, at 60°C for 5 s, and at 72°C for 10 s; and then

followed by a final step of fluorescence acquisition performed at 89°

C for 5 s.

Two standard curves constructed with 10-fold dilutions of synthetic

genomic and antigenomic HCV RNA (from 3.2 × 108 to 0.32 copies) were

used for the quantification of both HCV RNA stands. The specificity

of the assay for the detection of the antigenomic HCV RNA strand was

assessed by performing RT with the sense primer and serial dilutions

of the synthetic genomic HCV RNA as template. Linearity of the

quantification assay ranged from 3.2 to 3.2 × 108 copies of genomic

or antigenomic HCV RNA strand per reaction (figure 1). This assay was

capable of detecting 3.2 molecules of the correct strand while

unspecifically detecting 107-108 copies of the incorrect strand. The

sensitivity and dynamics of each assay were not affected when total

RNA extracted from HepG2 cells was added to the reaction.