Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

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Hepatitis C Virus Replicates in the Liver of Patients Who Have a

Sustained Response to Antiviral Treatment

Clinical Infectious Diseases Nov 15, 2006;43:1277-1283

Inmaculada Castillo, Elena Rodríguez-Iñigo, López-

Alcorocho, Margarita Pardo, Bartolomé, and Vicente Carreño

Foundation for the Study of Viral Hepatitis, Madrid, Spain

“….In summary, HCV persists and replicates in the livers and

PBMCs of a high percentage of patients who received antiviral

treatment for years after normalization of liver enzyme levels and

clearance of serum HCV RNA. Although it may be suspected that the

risk of HCV reactivation is smaller than the risk of hepatitis B

virus reactivation [13], persistence of HCV in these patients should

be taken into account under special circumstances (e.g.,

immunosuppression or chemotherapy). For example, there is a report of

the case of a single patient with chronic hepatitis C who experienced

serum HCV RNA clearance with normalization of aminotransferase

levels. After 8.5 years of test results that were negative for HCV

RNA, HCV infection reemerged following prednisone therapy [14].â€

ABSTRACT

Background. Positive-strand hepatitis C virus (HCV) RNA has been

detected in the livers of patients who have achieved a sustained

biochemical and virological response to antiviral therapy (hereafter,

referred to as sustained responders), but negative-strand HCV RNA was

undetectable in the hepatic tissue of these patients. We studied the

presence of both positive- and negative-strand HCV RNA in the livers

of 20 sustained responders with chronic hepatitis C whose response

persisted for a mean (± standard deviation [sD]) of 47.4 ± 32.8

months after treatment.

Methods. HCV RNA was tested by strand-specific, real-time reverse-

transcriptase polymerase chain reaction and by in situ hybridization

in posttreatment liver biopsy samples (obtained a mean [± SD] 35.4

± 35.0 months after therapy) and in patients' peripheral blood

mononuclear cells.

Results.

Positive-strand HCV RNA was found in 19 (95%) of 20 liver biopsy

specimens, and negative-strand HCV RNA was found in 15 (79%) of the

19 samples that had positive-strand HCV RNA. These results were

confirmed by in situ hybridization.

Regarding peripheral blood mononuclear cells, 13 (65%) of 20 samples

had positive-strand HCV RNA, and negative-strand HCV RNA was detected

in 12 (92%) of the 13 samples with positive-strand HCV RNA.

Liver necroinflammation was still present in the posttreatment liver

biopsy specimens of 15 patients, and fibrosis was present in 7,

although liver damage improved in all but 2 patients.

Conclusions. HCV persisted and replicated in the livers and

peripheral blood mononuclear cells of most sustained responders.

Thus, these patients did not experience HCV infection clearance,

despite apparent clinical disease resolution.

INTRODUCTION

Hepatitis C virus (HCV) infection represents a major health problem:

about 170 million people worldwide have chronic HCV infection. It is

thought that normalization of liver enzyme levels and loss of serum

HCV RNA, either after self-limited acute hepatitis C or after

successful antiviral therapy in chronically infected patients,

indicates clearance of the virus from the liver and, therefore,

resolution of infection. However, recent data suggest that HCV

infection may exist in the absence of viremia. We previously reported

that HCV can infect and replicate in the livers and PBMCs of patients

who have abnormal liver function values of unknown etiology in the

absence of anti-HCV and serum HCV RNA (called occult HCV infection)

[1, 2]. Similarly, Radkowski et al. [3] reported that HCV RNA

persisted in the livers of 3 of 11 patients with chronic hepatitis C

who had achieved a sustained biochemical and virological response

after antiviral therapy. However, negative-strand HCV RNA (the viral

replicative intermediate) was undetectable in the livers of these

patients.

Because of the important clinical implications of this finding, we

studied the presence of HCV RNA in the liver biopsy specimens of 20

patients with a sustained biochemical and virological response

(hereafter, referred to as sustained responders) in an attempt to

confirm and expand on those results. Special emphasis has been placed

on the study of the possible presence of negative-strand HCV RNA and,

thus, on HCV replication.

PATIENTS AND METHODS

Twenty patients with chronic hepatitis C who responded to antiviral

therapy were retrospectively included in this study. Patients had

been treated thrice weekly with 3 MU of recombinant a-IFN alone (6

patients) or in combination with 1000-1200 mg of ribavirin (4

patients) for 48 weeks, or they were treated once weekly with 1.5 ug

per kg of body weight with pegylated IFN alone (2 patients) or in

combination with 1000-1200 mg of ribavirin (8 patients) for 24 or 48

weeks. Patients were selected on the basis of the availability of a

stored posttreatment liver biopsy specimen and a PBMC sample obtained

the same day as the liver biopsy. They were also selected on the

basis of persistently normal liver function test results and negative

serum HCV RNA test results that remained for at least 16 months after

treatment. These 20 patients were contacted and gave their consent

for specific testing for HCV RNA in their stored posttreatment liver

biopsy and PBMC samples. The study was performed in accordance with

the principles of the Declaration of Helsinki.

All patients had histologically proven chronic hepatitis C before

treatment, all were anti-HCV and serum HCV RNA positive, and all had

abnormal liver enzyme values. Other causes of liver disease were

excluded; all patients were HIV-uninfected. The mean age (± SD) at

treatment was 42.7 ± 12.6 years, 14 patients (70%) were male, 17

(85%) were infected with HCV genotype 1b, and 3 (15%) were infected

with genotypes 2, 3, or 4. During the posttreatment follow-up (mean

[± SD] duration, 47.4 ± 32.8 months), liver function tests were

performed and serum HCV RNA was checked (Amplicor HCV v2.0; Roche

Diagnostics) every 6 months.

For all 20 patients, the posttreatment liver biopsy specimen was

obtained 35.4 ± 35.0 months after completion of therapy (range, 8-

117 months). The sample obtained during the liver biopsy was cut into

2 portions. One portion was used for histological examination and the

second was submerged (no later than 30 s after obtaining the liver

sample) in RNALater (Ambion) and stored at -20°C until use. PBMC

samples, obtained the same day as the liver biopsy and stored at -20°

C in RNALater (Ambion), were available for all patients. The pre- and

posttreatment liver biopsy samples were evaluated simultaneously by a

single pathologist and were assigned a Scheuer score [4] to compare

histological findings before and after therapy.

To assure the specificity of results, HCV RNA detection was performed

in a blinded fashion on different days by 2 different operators. PBMC

samples from 10 healthy, anti-HCV-negative subjects, total RNA

isolated from HepG2 cells, and blanks were used as negative controls.

Finally, all procedures were performed in accordance with the

recommendations of Kwok and Higuchi [5].

Quantitative, strand-specific, real-time RT-PCR. Total RNA was

isolated from liver and PBMC specimens using the SV Total RNA

Isolation kit (Promega), and the concentration was determined by

using spectrophotometric analysis.

Quantification of the 5' noncoding (5'NC) region of both HCV RNA

strands was performed as described elsewhere [6], using strand-

specific, real-time RT-PCR. The thermostable enzyme Tth was used at a

high temperature for the synthesis of cDNA. Briefly, for detecting

positive-strand HCV RNA, the cDNA was generated at 65°C for 20 min

in a 20-uL reaction mixture containing 0.5 ug of total RNA, 50 pmol/L

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

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

(Applied Biosystems) and 5 units of Tth (Applied Biosystems).

Thereafter, RT activity was inactivated by Mn2+ chelation with 8 L of

10× chelating buffer (Applied Biosystems), followed by heating at

95°C for 30 min. For detecting the negative-strand HCV RNA, the cDNA

was synthesized under the same conditions, but with the sense primer

UTRLC1 (5'-CTTCACGCAGAAAGCGTCTA-3'). Real-time RT-PCR was performed

in a LightCycler (Roche Molecular Biochemicals) with 2 uL of cDNA in

a final volume of 20 uL, using the LightCycler FastStart DNA Master

SYBR Green I kit (Roche Molecular Biochemicals). The reaction mixture

contained 4 mmol/L MgCl2, 0.5 umol/L of primers UTRLC1 and UTRLC2,

and 2 L of SYBR Green Master mix (Roche Molecular Biochemicals).

Amplification consisted of an initial denaturation and activation of

the enzyme for 10 min at 95°C, followed by 60 cycles at 95°C for 1

s, 60°C for 5 s, and 72°C for 10 s, and a final step of

fluorescence acquisition at 89°C for 5 s. Two standard curves with

synthetic HCV RNA of positive or negative polarity were constructed

for quantification of both HCV RNA strands. Linearity of the assay

ranged from 3.2 to 3.2 × 108 copies of positive-strand or negative-

strand HCV RNA per reaction. This assay detects 3.2 molecules of the

correct strand and nonspecifically detects 107-108 copies of the

incorrect strand [6]. Sensitivity and dynamics of each assay were not

affected when RNA isolated from HepG2 cells was added to the reaction.

Phylogenetic analysis. To guard against cross-contamination among

positive samples, the HCV core region was amplified from total RNA

isolated from the postreatment biopsy samples by RT-PCR, as described

elsewhere [1]. The 302-base pair core PCR products were cloned into

the pCRII-TOPO vector (Invitrogen), and 4 clones from each patient

were automatically sequenced. Sequences were aligned with core

sequences corresponding to all HCV genotypes retrieved from GenBank

using ClustalX, version 1.81 [7]. Phylogenetic and molecular

evolutionary analyses were conducted using Mega, version 2.1 [8].

Genetic distances were estimated using Kimura's 2-parameter method,

and SEs of distances were calculated using the bootstrap method (1000

replicates). A phylogenetic tree was constructed with the neighbor-

joining method, and its statistical significance was tested using the

bootstrap method (1000 replicates).

In situ hybridization. Paraffin-embedded liver sections (4 um)

were pretreated for in situ hybridization, as described elsewhere

[9]. Positive-strand HCV RNA was detected with a complementary RNA

probe obtained by in vitro transcription of the pC5' noncoding region

plasmid, which contains the complete 5' noncoding region of the HCV

genome. Negative-strand HCV RNA was detected with a complementary RNA

probe spanning 390 nucleotides of the HCV core coding region, which

was obtained by in vitro transcription of the pC core plasmid.

Hybridization conditions for detection of HCV RNA of both polarities

were described elsewhere [9]. The percentage of infected cells was

determined by visual inspection, and at least 2000 cells from each

liver section were counted.

Statistical analysis. Statistical analysis was done using SPSS

software, version 9.0 (SPSS). Continuous variables were expressed as

mean ± SD, except when otherwise indicated. Means were compared

using the Mann-Whitney U test, and paired data were compared using

the Wilcoxon signed rank test. Correlations were performed using

Spearman's test. A 2-sided P value <.05 was considered to be

statistically significant.

RESULTS

During the posttreatment follow-up (47.4 ± 32.8 months), liver

function tests (measuring aspartate aminotransferase, alanine

aminotransferase, and y-glutamyl transpeptidase levels) had

persistently normal results, and serum HCV RNA test results were

always negative.

Positive-strand HCV RNA was found in 19 (95%) of 20 liver biopsy

samples that were obtained 35.4 ± 35.0 months after the end of

treatment, and all negative controls were negative. The results of

HCV RNA detection performed by different operators on different days

were identical in all cases. The mean positive-strand HCV RNA load

(± SEM) was 1.9 × 105 ± 5.4 × 104 copies per ug of total RNA. The

presence of this intrahepatic positive-strand HCV RNA was confirmed

in all cases by in situ hybridization, and the percent of HCV-

infected hepatocytes was 4.5% ± 3.7%. The only liver biopsy sample

with undetectable viral RNA by strand-specific, real-time RT-PCR also

had negative results by in situ hybridization.

Negative-strand HCV RNA was found by strand-specific, real-time RT-

PCR in 15 (79%) of the 19 patients with intrahepatic positive-strand

HCV RNA. The mean negative-strand HCV RNA load (± SEM) was 7.3 ×

104 ± 3.1 × 104 copies per ug of total RNA, which is significantly

lower than that of positive-strand HCV RNA (P = .001). The only

patient whose biopsy sample did not yield intrahepatic positive-

strand HCV RNA also had negative results for negative-strand HCV RNA.

The presence of the negative strand was confirmed in all cases by in

situ hybridization. The percent of hepatocytes with hybridization

signals for negative-strand HCV RNA was 3.0% ± 2.6%, which was

significantly lower than the percent of hepatocytes with positive-

strand HCV RNA (P = .003). Individual data on HCV RNA detection are

shown in table 1.

HCV core sequencing was performed for 9 patients (7 with HCV genotype

1b infection, 1 with genotype 2 infection, and 1 with genotype 3

infection). In the remaining 10 patients with intrahepatic viral RNA,

it was not possible to perform core amplification, because RNA

samples were exhausted. The phylogenetic analysis demonstrated that

the genetic distances among clones from each patient were lower than

genetic distances among patients (table 2). The phylogenetic tree

shows that the clones were segregated in the 9 patients, indicating

that no cross-contamination occurred (figure 1). These results are in

agreement with the HCV genotypes that these 9 patients presented in

serum before treatment (table 1), because the sequences of the clones

from the patients were distributed together with the sequences of

their corresponding HCV genotype (1b, 2, or 3).

Positive-strand HCV RNA was detected in 13 (65%) of 20 PBMC samples

obtained on the same day that the posttreatment liver biopsy was

performed. The patient in whom HCV RNA was not detected in the liver

also did not have HCV detected in his PBMC sample. Negative-strand

HCV RNA was found in the PBMC samples of 12 (92%) of 13 persons with

HCV infection (table 1), and the mean (± SEM) HCV RNA load (1.6 ×

105 ± 5.5 × 104 copies per ug of total RNA) was significantly lower

than that of the positive-strand HCV RNA (9.5 × 105 ± 6.9 × 105

copies per ug of total RNA) (P = .012). When the amounts of HCV RNA

detected in the liver and PBMC samples were compared, we found that

the positive-strand HCV RNA load was significantly higher in PBMCs

than in liver specimens (mean [± SEM] viral load, 9.5 × 105 ± 6.9

× 105 copies per ug of total RNA vs. 1.9 × 105 ± 5.4 × 104 copies

per ug of total RNA; P = .034). Negative-strand HCV RNA load was also

significantly higher in PBMCs, compared with livers (mean [± SEM],

1.6 × 105 ± 5.5 × 104 copies per ug of total RNA vs. 7.3 × 104 ±

3.1 × 104 copies per ug of total RNA; P = .037). However, levels of

positive- or negative-strand HCV RNA in liver or PBMC samples and

time from treatment were not correlated (data not shown).

Regarding histological examination of the liver, there was a

significant improvement in necroinflammatory activity (portal plus

lobular inflammation), compared with the pretreatment values (score,

3.8 ± 1.6 vs. 1.3 ± 0.9; P < .001). Moreover, the fibrosis score

was significantly lower (P = .015) in the post-treatment liver biopsy

samples (0.7 ± 1.1) than in the pretreatment ones (1.2 ± 1.0).

Nevertheless, necroinflammation was still present in 15 patients, and

fibrosis was present in 7, although liver damage improved in all but

2 patients (table 3). Finally, no correlation was found between

necroinflammation in the posttreatment liver biopsy samples and

levels of positive- or negative-strand HCV RNA (data not shown).

DISCUSSION

We analyzed the presence of positive- and negative-strand HCV RNA in

the liver biopsy specimens of 20 sustained responders. Using strand-

specific, real-time RT-PCR, positive-strand HCV RNA was detected in

19 (95%) of the 20 liver biopsy specimens that were analyzed. The

specificity of the detection of the viral genome was confirmed by in

situ hybridization and by the HCV core sequence analysis showing that

there was no cross-contamination among liver samples. Negative-strand

HCV RNA (the replicative intermediate) was found (both by real-time

RT-PCR and by in situ hybridization) in 15 (79%) of the 19 liver

biopsy samples that had positive-strand HCV RNA. This indicates that

an ongoing viral replication was taking place in the livers of these

patients, which explains the persistence of intrahepatic HCV years

after a successful antiviral therapy outcome. The differences in the

antiviral treatments received by patients, as well as the difference

in the dates of the performance of the posttreatment liver biopsies

(range, 8-117 months), support the fact that HCV persistence is

unrelated to a specific treatment or to time since the end of

treatment.

We previously reported the presence and replication of HCV not only

in the livers of patients who had abnormal liver function values of

unknown etiology [1], but also in the livers of healthy anti-HCV-

positive patients who were serum HCV RNA negative and who had

persistently normal liver function test results [6]. The patients in

the present study comprise a different population, because they were

patients with chronic hepatitis C who responded to antiviral

treatment. Thus, occult HCV infection may be present in different

clinical situations.

Radkowski et al. [3] reported persistence of HCV in sustained

responders. Our results differ from those reported by these authors

in both the percentage of patients with positive-strand HCV RNA

detected in their livers (95% vs. 27%), as well as in the detection

of viral replication (79% vs. 0%). There are 2 possible explanations

for these discrepancies. First, in our study, liver biopsies were

performed a mean (±SD) of 35.3 ± 35.0 months after the end of

treatment, and Radkowski et al. [3] included liver biopsies performed

63.6 ± 16.7 months after therapy. Thus, intrahepatic levels of

positive- and negative-strand HCV RNA might decrease over time,

becoming undetectable in sustained responders. However, there are

some data that do not support this hypothesis. We have detected the

positive- and negative-strand HCV RNA in liver biopsy samples

obtained 50 months after treatment, whereas Radkowski et al. [3] did

not find HCV RNA in 4 liver biopsy samples obtained 41, 50, 54, and

55 months after therapy. In addition, 1 of our liver biopsy samples

with positive- and negative-strand HCV RNA was obtained 117 months

after treatment, which is a longer posttreatment period than that

experienced by the patients of Radkowski et al. [3] (maximum length

of follow-up, 98 months). Another possibility may lie in the

integrity of the intrahepatic HCV RNA. It has been established that

the time elapsed between obtaining and freezing the liver biopsy

specimen (to inhibit intracellular RNAse) must be no longer than 3

min to prevent RNA degradation and to ensure detection of positive-

strand HCV RNA and especially negative-strand HCV RNA [10]. In the

present study, liver biopsy specimens were submerged in a chemical

agent that inhibits RNAses within 30 s after they were obtaine to

preserve the integrity of RNA, but information about measures taken

to preserve RNA integrity was not provided by Radkowski et al. [3].

The mean ratio of positive- to negative-strand HCV RNA found in the

livers of our sustained responders was 2.6 : 1, differing from the

data of a previous study that also used a quantitative, strand-

specific, real-time PCR and reported ratios of 100 : 1-1000 : 1 [11].

However, although our patients were serum HCV RNA negative, the study

by Pradel-Komurian [11] analyzed viral RNA strands in the livers of

chronic hepatitis C patients with HCV RNA in serum. Thus,

contamination by circulating virions (that contain positive-strand

HCV RNA) cannot be discarded, and this contributes to those high

ratios. Moreover, positive strand to negative strand ratios could be

affected by HCV RNA degradation if liver biopsy samples are not

properly stored, because the decrease in the titer of negative-strand

HCV RNA is more pronounced than that of positive-strand HCV RNA [10].

PBMC samples obtained on the same day the liver biopsies were

performed were available from all patients, and positive-strand HCV

RNA was detected in 13 (65%) of them. Negative-strand HCV RNA was

also found in 12 (92%) of the 13 PBMC samples harboring positive-

strand HCV RNA. The mean ratio of positive to negative strands in the

PBMC samples of our patients was 5.9, which is similar to the mean

ratio of 6.6 reported elsewhere in lymphocytes and macrophages of

sustained responders [3]. Thus, our findings agree with previous

studies, except that we detected HCV replication without exogenous

cell activation, and in other reports, negative-strand HCV RNA was

detected only after mitogen stimulation of PBMCs [3, 12]. We do not

have a clear explanation for this difference. One possible

explanation could be that HCV genotypes infecting our patients were

different than those found in the other mentioned reports. However,

this was not true; most patients in all of these studies were

infected with HCV genotype 1. We also found that the loads of

positive- and negative-strand HCV RNA in our patients were

significantly higher in PBMC samples than in liver biopsy samples,

which suggests that the HCV strain detected in our sustained

responders replicates more efficiently in PBMCs than in livers.

Finally, regarding histological damage present in posttreatment liver

biopsy samples, it should be stated that 15 patients had continued

liver necroinflammation, and 1 patient had liver fibrosis; however,

there was an overall improvement in histological damage. So, it is

difficult to know whether persistence of HCV infection and

replication has a clinical relevance until more data become available.

In summary, HCV persists and replicates in the livers and PBMCs of a

high percentage of patients who received antiviral treatment for

years after normalization of liver enzyme levels and clearance of

serum HCV RNA. Although it may be suspected that the risk of HCV

reactivation is smaller than the risk of hepatitis B virus

reactivation [13], persistence of HCV in these patients should be

taken into account under special circumstances (e.g.,

immunosuppression or chemotherapy). For example, there is a report of

the case of a single patient with chronic hepatitis C who experienced

serum HCV RNA clearance with normalization of aminotransferase

levels. After 8.5 years of test results that were negative for HCV

RNA, HCV infection reemerged following prednisone therapy [14].

[Guest guest]

THIS is EXACTLY why I refuse to take Prednisone or any other immunosuppressant!elizabethnv1 <elizabethnv1@...> wrote: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283Inmaculada Castillo, Elena Rodríguez-Iñigo, López-Alcorocho, Margarita Pardo, Bartolomé, and Vicente CarreñoFoundation for the Study of Viral Hepatitis, Madrid,

Spain“….In summary, HCV persists and replicates in the livers and PBMCs of a high percentage of patients who received antiviral treatment for years after normalization of liver enzyme levels and clearance of serum HCV RNA. Although it may be suspected that the risk of HCV reactivation is smaller than the risk of hepatitis B virus reactivation [13], persistence of HCV in these patients should be taken into account under special circumstances (e.g., immunosuppression or chemotherapy). For example, there is a report of the case of a single patient with chronic hepatitis C who experienced serum HCV RNA clearance with normalization of aminotransferase levels. After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].â€ABSTRACTBackground. Positive-strand hepatitis C virus (HCV) RNA has been detected in the livers of patients who have

achieved a sustained biochemical and virological response to antiviral therapy (hereafter, referred to as sustained responders), but negative-strand HCV RNA was undetectable in the hepatic tissue of these patients. We studied the presence of both positive- and negative-strand HCV RNA in the livers of 20 sustained responders with chronic hepatitis C whose response persisted for a mean (± standard deviation [sD]) of 47.4 ± 32.8 months after treatment.Methods. HCV RNA was tested by strand-specific, real-time reverse-transcriptase polymerase chain reaction and by in situ hybridization in posttreatment liver biopsy samples (obtained a mean [± SD] 35.4 ± 35.0 months after therapy) and in patients' peripheral blood mononuclear cells.Results. Positive-strand HCV RNA was found in 19 (95%) of 20 liver biopsy specimens, and negative-strand HCV RNA was found in 15 (79%) of the 19 samples that had

positive-strand HCV RNA. These results were confirmed by in situ hybridization. Regarding peripheral blood mononuclear cells, 13 (65%) of 20 samples had positive-strand HCV RNA, and negative-strand HCV RNA was detected in 12 (92%) of the 13 samples with positive-strand HCV RNA. Liver necroinflammation was still present in the posttreatment liver biopsy specimens of 15 patients, and fibrosis was present in 7, although liver damage improved in all but 2 patients.Conclusions. HCV persisted and replicated in the livers and peripheral blood mononuclear cells of most sustained responders. Thus, these patients did not experience HCV infection clearance, despite apparent clinical disease resolution.INTRODUCTIONHepatitis C virus (HCV) infection represents a major health problem: about 170 million people worldwide have chronic HCV infection. It is thought that normalization of liver enzyme levels and loss of

serum HCV RNA, either after self-limited acute hepatitis C or after successful antiviral therapy in chronically infected patients, indicates clearance of the virus from the liver and, therefore, resolution of infection. However, recent data suggest that HCV infection may exist in the absence of viremia. We previously reported that HCV can infect and replicate in the livers and PBMCs of patients who have abnormal liver function values of unknown etiology in the absence of anti-HCV and serum HCV RNA (called occult HCV infection) [1, 2]. Similarly, Radkowski et al. [3] reported that HCV RNA persisted in the livers of 3 of 11 patients with chronic hepatitis C who had achieved a sustained biochemical and virological response after antiviral therapy. However, negative-strand HCV RNA (the viral replicative intermediate) was undetectable in the livers of these patients.Because of the important clinical implications of

this finding, we studied the presence of HCV RNA in the liver biopsy specimens of 20 patients with a sustained biochemical and virological response (hereafter, referred to as sustained responders) in an attempt to confirm and expand on those results. Special emphasis has been placed on the study of the possible presence of negative-strand HCV RNA and, thus, on HCV replication.PATIENTS AND METHODSTwenty patients with chronic hepatitis C who responded to antiviral therapy were retrospectively included in this study. Patients had been treated thrice weekly with 3 MU of recombinant a-IFN alone (6 patients) or in combination with 1000-1200 mg of ribavirin (4 patients) for 48 weeks, or they were treated once weekly with 1.5 ug per kg of body weight with pegylated IFN alone (2 patients) or in combination with 1000-1200 mg of ribavirin (8 patients) for 24 or 48 weeks. Patients were selected on the basis of the

availability of a stored posttreatment liver biopsy specimen and a PBMC sample obtained the same day as the liver biopsy. They were also selected on the basis of persistently normal liver function test results and negative serum HCV RNA test results that remained for at least 16 months after treatment. These 20 patients were contacted and gave their consent for specific testing for HCV RNA in their stored posttreatment liver biopsy and PBMC samples. The study was performed in accordance with the principles of the Declaration of Helsinki.All patients had histologically proven chronic hepatitis C before treatment, all were anti-HCV and serum HCV RNA positive, and all had abnormal liver enzyme values. Other causes of liver disease were excluded; all patients were HIV-uninfected. The mean age (± SD) at treatment was 42.7 ± 12.6 years, 14 patients (70%) were male, 17 (85%) were infected with HCV genotype 1b, and 3

(15%) were infected with genotypes 2, 3, or 4. During the posttreatment follow-up (mean [± SD] duration, 47.4 ± 32.8 months), liver function tests were performed and serum HCV RNA was checked (Amplicor HCV v2.0; Roche Diagnostics) every 6 months.For all 20 patients, the posttreatment liver biopsy specimen was obtained 35.4 ± 35.0 months after completion of therapy (range, 8-117 months). The sample obtained during the liver biopsy was cut into 2 portions. One portion was used for histological examination and the second was submerged (no later than 30 s after obtaining the liver sample) in RNALater (Ambion) and stored at -20°C until use. PBMC samples, obtained the same day as the liver biopsy and stored at -20°C in RNALater (Ambion), were available for all patients. The pre- and posttreatment liver biopsy samples were evaluated simultaneously by a single pathologist and were assigned a Scheuer score [4] to

compare histological findings before and after therapy.To assure the specificity of results, HCV RNA detection was performed in a blinded fashion on different days by 2 different operators. PBMC samples from 10 healthy, anti-HCV-negative subjects, total RNA isolated from HepG2 cells, and blanks were used as negative controls. Finally, all procedures were performed in accordance with the recommendations of Kwok and Higuchi [5].Quantitative, strand-specific, real-time RT-PCR. Total RNA was isolated from liver and PBMC specimens using the SV Total RNA Isolation kit (Promega), and the concentration was determined by using spectrophotometric analysis.Quantification of the 5' noncoding (5'NC) region of both HCV RNA strands was performed as described elsewhere [6], using strand-specific, real-time RT-PCR. The thermostable enzyme Tth was used at a high temperature for the synthesis of cDNA. Briefly, for

detecting positive-strand HCV RNA, the cDNA was generated at 65°C for 20 min in a 20-uL reaction mixture containing 0.5 ug of total RNA, 50 pmol/L of antisense primer UTRLC2 (5'-CAAGCACCCTATCAGGCAGT-3'), 1 mmol/L MnCl2, 200 mol/L of each deoxynucleotide triphosphate, 1× RT buffer (Applied Biosystems) and 5 units of Tth (Applied Biosystems). Thereafter, RT activity was inactivated by Mn2+ chelation with 8 L of 10× chelating buffer (Applied Biosystems), followed by heating at 95°C for 30 min. For detecting the negative-strand HCV RNA, the cDNA was synthesized under the same conditions, but with the sense primer UTRLC1 (5'-CTTCACGCAGAAAGCGTCTA-3'). Real-time RT-PCR was performed in a LightCycler (Roche Molecular Biochemicals) with 2 uL of cDNA in a final volume of 20 uL, using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals). The reaction mixture contained

4 mmol/L MgCl2, 0.5 umol/L of primers UTRLC1 and UTRLC2, and 2 L of SYBR Green Master mix (Roche Molecular Biochemicals). Amplification consisted of an initial denaturation and activation of the enzyme for 10 min at 95°C, followed by 60 cycles at 95°C for 1 s, 60°C for 5 s, and 72°C for 10 s, and a final step of fluorescence acquisition at 89°C for 5 s. Two standard curves with synthetic HCV RNA of positive or negative polarity were constructed for quantification of both HCV RNA strands. Linearity of the assay ranged from 3.2 to 3.2 × 108 copies of positive-strand or negative-strand HCV RNA per reaction. This assay detects 3.2 molecules of the correct strand and nonspecifically detects 107-108 copies of the incorrect strand [6]. Sensitivity and dynamics of each assay were not affected when RNA isolated from HepG2 cells was added to the reaction.Phylogenetic analysis. To guard against cross-contamination

among positive samples, the HCV core region was amplified from total RNA isolated from the postreatment biopsy samples by RT-PCR, as described elsewhere [1]. The 302-base pair core PCR products were cloned into the pCRII-TOPO vector (Invitrogen), and 4 clones from each patient were automatically sequenced. Sequences were aligned with core sequences corresponding to all HCV genotypes retrieved from GenBank using ClustalX, version 1.81 [7]. Phylogenetic and molecular evolutionary analyses were conducted using Mega, version 2.1 [8]. Genetic distances were estimated using Kimura's 2-parameter method, and SEs of distances were calculated using the bootstrap method (1000 replicates). A phylogenetic tree was constructed with the neighbor-joining method, and its statistical significance was tested using the bootstrap method (1000 replicates).In situ hybridization. Paraffin-embedded liver sections (4 um) were

pretreated for in situ hybridization, as described elsewhere [9]. Positive-strand HCV RNA was detected with a complementary RNA probe obtained by in vitro transcription of the pC5' noncoding region plasmid, which contains the complete 5' noncoding region of the HCV genome. Negative-strand HCV RNA was detected with a complementary RNA probe spanning 390 nucleotides of the HCV core coding region, which was obtained by in vitro transcription of the pC core plasmid. Hybridization conditions for detection of HCV RNA of both polarities were described elsewhere [9]. The percentage of infected cells was determined by visual inspection, and at least 2000 cells from each liver section were counted.Statistical analysis. Statistical analysis was done using SPSS software, version 9.0 (SPSS). Continuous variables were expressed as mean ± SD, except when otherwise indicated. Means were compared using the Mann-Whitney U test,

and paired data were compared using the Wilcoxon signed rank test. Correlations were performed using Spearman's test. A 2-sided P value <.05 was considered to be statistically significant.RESULTSDuring the posttreatment follow-up (47.4 ± 32.8 months), liver function tests (measuring aspartate aminotransferase, alanine aminotransferase, and y-glutamyl transpeptidase levels) had persistently normal results, and serum HCV RNA test results were always negative.Positive-strand HCV RNA was found in 19 (95%) of 20 liver biopsy samples that were obtained 35.4 ± 35.0 months after the end of treatment, and all negative controls were negative. The results of HCV RNA detection performed by different operators on different days were identical in all cases. The mean positive-strand HCV RNA load (± SEM) was 1.9 × 105 ± 5.4 × 104 copies per ug of total RNA. The presence of this intrahepatic

positive-strand HCV RNA was confirmed in all cases by in situ hybridization, and the percent of HCV-infected hepatocytes was 4.5% ± 3.7%. The only liver biopsy sample with undetectable viral RNA by strand-specific, real-time RT-PCR also had negative results by in situ hybridization.Negative-strand HCV RNA was found by strand-specific, real-time RT-PCR in 15 (79%) of the 19 patients with intrahepatic positive-strand HCV RNA. The mean negative-strand HCV RNA load (± SEM) was 7.3 × 104 ± 3.1 × 104 copies per ug of total RNA, which is significantly lower than that of positive-strand HCV RNA (P = .001). The only patient whose biopsy sample did not yield intrahepatic positive-strand HCV RNA also had negative results for negative-strand HCV RNA. The presence of the negative strand was confirmed in all cases by in situ hybridization. The percent of hepatocytes with hybridization signals for negative-strand HCV RNA

was 3.0% ± 2.6%, which was significantly lower than the percent of hepatocytes with positive-strand HCV RNA (P = .003). Individual data on HCV RNA detection are shown in table 1.HCV core sequencing was performed for 9 patients (7 with HCV genotype 1b infection, 1 with genotype 2 infection, and 1 with genotype 3 infection). In the remaining 10 patients with intrahepatic viral RNA, it was not possible to perform core amplification, because RNA samples were exhausted. The phylogenetic analysis demonstrated that the genetic distances among clones from each patient were lower than genetic distances among patients (table 2). The phylogenetic tree shows that the clones were segregated in the 9 patients, indicating that no cross-contamination occurred (figure 1). These results are in agreement with the HCV genotypes that these 9 patients presented in serum before treatment (table 1), because the sequences of the clones

from the patients were distributed together with the sequences of their corresponding HCV genotype (1b, 2, or 3).Positive-strand HCV RNA was detected in 13 (65%) of 20 PBMC samples obtained on the same day that the posttreatment liver biopsy was performed. The patient in whom HCV RNA was not detected in the liver also did not have HCV detected in his PBMC sample. Negative-strand HCV RNA was found in the PBMC samples of 12 (92%) of 13 persons with HCV infection (table 1), and the mean (± SEM) HCV RNA load (1.6 × 105 ± 5.5 × 104 copies per ug of total RNA) was significantly lower than that of the positive-strand HCV RNA (9.5 × 105 ± 6.9 × 105 copies per ug of total RNA) (P = .012). When the amounts of HCV RNA detected in the liver and PBMC samples were compared, we found that the positive-strand HCV RNA load was significantly higher in PBMCs than in liver specimens (mean [± SEM] viral load, 9.5 × 105 ± 6.9

× 105 copies per ug of total RNA vs. 1.9 × 105 ± 5.4 × 104 copies per ug of total RNA; P = .034). Negative-strand HCV RNA load was also significantly higher in PBMCs, compared with livers (mean [± SEM], 1.6 × 105 ± 5.5 × 104 copies per ug of total RNA vs. 7.3 × 104 ± 3.1 × 104 copies per ug of total RNA; P = .037). However, levels of positive- or negative-strand HCV RNA in liver or PBMC samples and time from treatment were not correlated (data not shown).Regarding histological examination of the liver, there was a significant improvement in necroinflammatory activity (portal plus lobular inflammation), compared with the pretreatment values (score, 3.8 ± 1.6 vs. 1.3 ± 0.9; P < .001). Moreover, the fibrosis score was significantly lower (P = .015) in the post-treatment liver biopsy samples (0.7 ± 1.1) than in the pretreatment ones (1.2 ± 1.0). Nevertheless, necroinflammation was still present

in 15 patients, and fibrosis was present in 7, although liver damage improved in all but 2 patients (table 3). Finally, no correlation was found between necroinflammation in the posttreatment liver biopsy samples and levels of positive- or negative-strand HCV RNA (data not shown).DISCUSSIONWe analyzed the presence of positive- and negative-strand HCV RNA in the liver biopsy specimens of 20 sustained responders. Using strand-specific, real-time RT-PCR, positive-strand HCV RNA was detected in 19 (95%) of the 20 liver biopsy specimens that were analyzed. The specificity of the detection of the viral genome was confirmed by in situ hybridization and by the HCV core sequence analysis showing that there was no cross-contamination among liver samples. Negative-strand HCV RNA (the replicative intermediate) was found (both by real-time RT-PCR and by in situ hybridization) in 15 (79%) of the 19 liver biopsy samples

that had positive-strand HCV RNA. This indicates that an ongoing viral replication was taking place in the livers of these patients, which explains the persistence of intrahepatic HCV years after a successful antiviral therapy outcome. The differences in the antiviral treatments received by patients, as well as the difference in the dates of the performance of the posttreatment liver biopsies (range, 8-117 months), support the fact that HCV persistence is unrelated to a specific treatment or to time since the end of treatment.We previously reported the presence and replication of HCV not only in the livers of patients who had abnormal liver function values of unknown etiology [1], but also in the livers of healthy anti-HCV-positive patients who were serum HCV RNA negative and who had persistently normal liver function test results [6]. The patients in the present study comprise a different population, because they

were patients with chronic hepatitis C who responded to antiviral treatment. Thus, occult HCV infection may be present in different clinical situations.Radkowski et al. [3] reported persistence of HCV in sustained responders. Our results differ from those reported by these authors in both the percentage of patients with positive-strand HCV RNA detected in their livers (95% vs. 27%), as well as in the detection of viral replication (79% vs. 0%). There are 2 possible explanations for these discrepancies. First, in our study, liver biopsies were performed a mean (±SD) of 35.3 ± 35.0 months after the end of treatment, and Radkowski et al. [3] included liver biopsies performed 63.6 ± 16.7 months after therapy. Thus, intrahepatic levels of positive- and negative-strand HCV RNA might decrease over time, becoming undetectable in sustained responders. However, there are some data that do not support this hypothesis.

We have detected the positive- and negative-strand HCV RNA in liver biopsy samples obtained 50 months after treatment, whereas Radkowski et al. [3] did not find HCV RNA in 4 liver biopsy samples obtained 41, 50, 54, and 55 months after therapy. In addition, 1 of our liver biopsy samples with positive- and negative-strand HCV RNA was obtained 117 months after treatment, which is a longer posttreatment period than that experienced by the patients of Radkowski et al. [3] (maximum length of follow-up, 98 months). Another possibility may lie in the integrity of the intrahepatic HCV RNA. It has been established that the time elapsed between obtaining and freezing the liver biopsy specimen (to inhibit intracellular RNAse) must be no longer than 3 min to prevent RNA degradation and to ensure detection of positive-strand HCV RNA and especially negative-strand HCV RNA [10]. In the present study, liver biopsy specimens were

submerged in a chemical agent that inhibits RNAses within 30 s after they were obtaine to preserve the integrity of RNA, but information about measures taken to preserve RNA integrity was not provided by Radkowski et al. [3].The mean ratio of positive- to negative-strand HCV RNA found in the livers of our sustained responders was 2.6 : 1, differing from the data of a previous study that also used a quantitative, strand-specific, real-time PCR and reported ratios of 100 : 1-1000 : 1 [11]. However, although our patients were serum HCV RNA negative, the study by Pradel-Komurian [11] analyzed viral RNA strands in the livers of chronic hepatitis C patients with HCV RNA in serum. Thus, contamination by circulating virions (that contain positive-strand HCV RNA) cannot be discarded, and this contributes to those high ratios. Moreover, positive strand to negative strand ratios could be affected by HCV RNA degradation if

liver biopsy samples are not properly stored, because the decrease in the titer of negative-strand HCV RNA is more pronounced than that of positive-strand HCV RNA [10].PBMC samples obtained on the same day the liver biopsies were performed were available from all patients, and positive-strand HCV RNA was detected in 13 (65%) of them. Negative-strand HCV RNA was also found in 12 (92%) of the 13 PBMC samples harboring positive-strand HCV RNA. The mean ratio of positive to negative strands in the PBMC samples of our patients was 5.9, which is similar to the mean ratio of 6.6 reported elsewhere in lymphocytes and macrophages of sustained responders [3]. Thus, our findings agree with previous studies, except that we detected HCV replication without exogenous cell activation, and in other reports, negative-strand HCV RNA was detected only after mitogen stimulation of PBMCs [3, 12]. We do not have a clear explanation for

this difference. One possible explanation could be that HCV genotypes infecting our patients were different than those found in the other mentioned reports. However, this was not true; most patients in all of these studies were infected with HCV genotype 1. We also found that the loads of positive- and negative-strand HCV RNA in our patients were significantly higher in PBMC samples than in liver biopsy samples, which suggests that the HCV strain detected in our sustained responders replicates more efficiently in PBMCs than in livers.Finally, regarding histological damage present in posttreatment liver biopsy samples, it should be stated that 15 patients had continued liver necroinflammation, and 1 patient had liver fibrosis; however, there was an overall improvement in histological damage. So, it is difficult to know whether persistence of HCV infection and replication has a clinical relevance until more data

become available.In summary, HCV persists and replicates in the livers and PBMCs of a high percentage of patients who received antiviral treatment for years after normalization of liver enzyme levels and clearance of serum HCV RNA. Although it may be suspected that the risk of HCV reactivation is smaller than the risk of hepatitis B virus reactivation [13], persistence of HCV in these patients should be taken into account under special circumstances (e.g., immunosuppression or chemotherapy). For example, there is a report of the case of a single patient with chronic hepatitis C who experienced serum HCV RNA clearance with normalization of aminotransferase levels. After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].Jackie

[Guest guest]

Hepatitis C Virus Replicates in the Liver of Patients Who Have a

Sustained Response to Antiviral Treatment

Clinical Infectious Diseases Nov 15, 2006;43:1277-1283

..............After 8.5 years of test results that were negative for HCV

RNA, HCV infection reemerged following prednisone therapy [14].

(Nikki)

So it appears that the virus does not really clear, just goes deep into

hiding?

[Guest guest]

It goes in to remission, does not replicate the virus just stays dormant. If the virus count gets down to non detectable level , but the virus is always there. Janet Nikki Cowan <nikkicowan@...> wrote: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15,

2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?Take the ordinary things of life, and make them your own. Do the impossible with a smile

[Guest guest]

Yes , but that does not mean the person infected will relapse . So far those that have a sustained viralogical response to the treatment for over 6months are staying clear and undectable . But they must always be aware of the fact that it can come back at any time . Its kinda like a person who goes through chemo for cancer and the cancer goes into remission , but it can always decide to come back years later . So living healthy and staying away from drugs and alcahol help to prevent relapse

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

[Guest guest]

That it true Ally and its really sad,, to have fought to put this virus into remission and then give it the opportunity to reawaken... sad and stupid.. but some ppl just do not get it that they really shouldnt drink as some ppl DO have problems with alcohol and addiction issues and the alcohol is an immune suppressant, just like prednisone.. Ally <4thMoon@...> wrote: And the sad fact is that so many people who do clear think that is an open invitation to drink then wonder why they have problems down the road...

sigh On 10/25/06, elizabethnv1 <elizabethnv1earthlink (DOT) net> wrote: Yes , but that does not mean the person infected will relapse . So far those that have a sustained viralogical response to the treatment for over 6months are staying clear and undectable . But they must always be aware of the fact that it can come back at any time . Its kinda like a person who goes through chemo for cancer and the cancer goes into remission , but it can always decide to come back years later . So living healthy and staying away from drugs and alcahol help to prevent relapse -----

Original Message ----- From: Nikki Cowan Hepatitis C Sent: Wednesday, October 25, 2006 7:06 AM Subject: RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV

infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding? Jackie

[Guest guest]

I'm curious... when it goes dormant (remission) can someone still catch it from you if they come in contact with your blood?

Ally

On 10/25/06, Jackie on <redjaxjm@...> wrote:

That it true Ally and its really sad,, to have fought to put this virus into remission and then give it the opportunity to reawaken... sad and stupid.. but some ppl just do not get it that they really shouldnt drink as some ppl DO have problems with alcohol and addiction issues and the alcohol is an immune suppressant, just like prednisone.. Ally <4thMoon@...> wrote:

And the sad fact is that so many people who do clear think that is an open invitation to drink then wonder why they have problems down the road... sigh

On 10/25/06, elizabethnv1 <elizabethnv1@...

> wrote:

Yes , but that does not mean the person infected will relapse . So far those that have a sustained viralogical response to the treatment for over 6months are staying clear and undectable . But they must always be aware of the fact that it can come back at any time . Its kinda like a person who goes through chemo for cancer and the cancer goes into remission , but it can always decide to come back years later . So living healthy and staying away from drugs and alcahol help to prevent relapse

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

[Guest guest]

Oh boy I never even thought to research that one , boy I must be getting slow . You have a really good questions there Ally , one that I am going to email my hepatologist about . Unless Jackie knows the answer , I am curious about it too ..

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

[Guest guest]

Right, I mean, I've always assumed that once you have it, that's it but maybe not. I'll have to ask my Dr too next time I talk to her.

Ally

On 10/26/06, elizabethnv1 <elizabethnv1@...> wrote:

Oh boy I never even thought to research that one , boy I must be getting slow . You have a really good questions there Ally , one that I am going to email my hepatologist about . Unless Jackie knows the answer , I am curious about it too ..

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

[Guest guest]

Lemme know what you find out , and I will also try to see what I can find out .

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

[Guest guest]

I dont know the answer to that one either Liz,, I would think that if you are undetectible, that its not transferrable but I honestly dont know,, so I hope whoever finds the answer out to this , lets us all know!elizabethnv1 <elizabethnv1@...> wrote: Lemme know what you find out , and I will also try to see what I can find out . RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding? Jackie Jackie

[Guest guest]

The more I have time to think about it , the more I believe the theory that if hcv is still present on a molecular level in a svr person then they can still infect others . That is if the blood to blood factor is there .

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

Jackie

[Guest guest]

I'd bet you are right,, if there is blood to blood contact!elizabethnv1 <elizabethnv1@...> wrote: The more I have time to think about it , the more I believe the theory that if hcv is still present on a molecular level in a svr person then they can still infect others . That is if the blood to blood factor is there . RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus

does not really clear, just goes deep intohiding? Jackie Jackie Jackie

[Guest guest]

Hi,

If Anti HCV is positive even

if the patient

has SVR, does this mean the virus

has not been completely cleared?

Regards

From: Hepatitis C [mailto:Hepatitis C ] On Behalf Of elizabethnv1

Sent: Friday, October 27, 2006 8:22 AM

Hepatitis C

Subject: Re:

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained

Respo

The more I have time to think about it ,

the more I believe the theory that if hcv is still present on a

molecular level in a svr person then they can still infect others . That is if

the blood to blood factor is there .

RE:

Hepatitis C Virus Replicates in the Liver of

Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a

Sustained Response to Antiviral Treatment

Clinical Infectious Diseases Nov 15, 2006;43:1277-1283

..............After 8.5 years of test results that

were negative for HCV

RNA, HCV infection reemerged following prednisone therapy [14].

(Nikki)

So it appears that the virus does not really clear, just goes deep into

hiding?

Jackie

Jackie

[Guest guest]

I am pretty sure about it , but will ask my doc to be sure .

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

Jackie

Jackie

[Guest guest]

Even if a person attains a sustained viralogical response there is still hcv present at the molecular level . Having a svr means that the virus is no longer growing , but it is still there nonetheless . It no longer is doing damage to the liver , but will always be there so keeping healthy is important .

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

Jackie

[Guest guest]

I know this. What I am

asking it is that ANTI HCV

is postive after teratment even if you have

SVR. Is it because HCV virus

present at molecular level?

From: Hepatitis C [mailto:Hepatitis C ] On Behalf Of elizabethnv1

Sent: Friday, October 27, 2006 9:33 AM

Hepatitis C

Subject: Re:

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained

Respo

Even if a person attains a sustained viralogical response

there is still hcv present at the molecular level .

Having a svr means that the virus is no longer growing , but

it is still there nonetheless . It no longer is doing damage to the liver , but will always be there so keeping healthy is

important .

RE:

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a

Sustained Response to Antiviral Treatment

Clinical Infectious Diseases Nov 15, 2006;43:1277-1283

..............After 8.5

years of test results that were negative for HCV

RNA, HCV infection reemerged following prednisone therapy [14].

(Nikki)

So it appears that the virus does not really clear, just goes deep into

hiding?

Jackie

Jackie

[Guest guest]

Yes Aylin, That is correct, The HCV remains present at the molecular level. That is why they will not take blood at blood center such as Red Cross because we effectively become carriers of the disease even tho it is not active in our bodies. Tis a shame too, because I have a type of hemoglobin that is indigenous to Native American Indians, the Red Cross used to beg for my blood because of this hemoglobin abnormally. Very few less than full blood indians have it. But what I can tell you it cause me to have more RBC then the average person. I will do some research on it to fully explain what I am talking about. Love JanetAylin KANTARCI <aylin.kantarci@...> wrote: I know this. What I am asking it is that ANTI HCV is postive after teratment even if you have SVR. Is it because HCV

virus present at molecular level? From: Hepatitis C [mailto:Hepatitis C ] On Behalf Of elizabethnv1Sent: Friday, October 27, 2006 9:33 AMHepatitis C Subject: Re: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo Even if a person attains a sustained viralogical response there is still hcv present at the molecular level . Having a svr means that the virus is no longer growing , but it is still there nonetheless . It no longer is doing damage to the liver ,

but will always be there so keeping healthy is important . RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding? Jackie

Jackie Take the ordinary

things of life, and make them your own. Do the impossible with a smile

[Guest guest]

Yes

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

Jackie

[Guest guest]

me too,, now I would think that the risk factor of tranmission would be somewhat less if you are undetectible for virus say down to the 2-5 iu/ml and if you had a real small occult scratch say on your hand and you got someone else's blood on your hand, you would have a risk of transmission but I would wonder IF IT would be LESS of a risk because of being undetectible.. thats different than giving blood and then someone receiving a unit of your blood,, that would be direct contact,, does this make sense? Im tired tonite, I did not sleep last nite...elizabethnv1 <elizabethnv1@...> wrote: I am pretty sure about it , but will ask my doc to be sure . RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding? Jackie Jackie Jackie Jackie

[Guest guest]

I have been down myself , and right now my head is a little fuzzy . I will reread this tomorrow when my brain wakes up ,lol

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

Jackie

Jackie

Jackie

[Guest guest]

I have been down myself , and right now my head is a little fuzzy . I will reread this tomorrow when my brain wakes up ,lol

RE: Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Respo

Hepatitis C Virus Replicates in the Liver of Patients Who Have a Sustained Response to Antiviral TreatmentClinical Infectious Diseases Nov 15, 2006;43:1277-1283.............After 8.5 years of test results that were negative for HCV RNA, HCV infection reemerged following prednisone therapy [14].(Nikki)So it appears that the virus does not really clear, just goes deep intohiding?

Jackie

Jackie

Jackie

Jackie