the brazilian journal of infectious diseases

Transcription

BJID 2007; 11 (October)
Volume 11
•
1
ISSN 1413-8670
Supplement 1
•
October 2007
THE BRAZILIAN JOURNAL OF
INFECTIOUS DISEASES
An Official Publication of the Brazilian Society of Infectious Diseases
EDITOR
Anastácio Q. Sousa
Consensus of the Brazilian Society of Infectious Diseases
on the Management and Treatment of Hepatitis C
PUBLISHED BY CONTEXTO
October 2007
Printed in Brazil
www.bjid.com.br
2
THE BRAZILIAN SOCIETY OF INFECTIOUS DISEASES
The Brazilian Society of Infectious Diseases is conducted for scientific purposes, for the advancement
and promulgation of knowledge relevant to infectious diseases.
OFFICERS
2007-2008
President
João Silva de Mendonça
Vice President
Denise Vantil Marangoni
First Secretary
Juvêncio José D. Furtado
Second Secretary
Érico Antônio Gomes de Arruda
First Treasurer
Roberto Marcio da Costa Florim
Second Treasurer
Flávio de Queiroz Telles Filho
Federal
Federal
Alagoas - Maria Raquel dos A.S. Guimarães
Amazonas - Eucides Batista da Silva
Bahia - Adriano Oliveira
Ceará - Anastácio de Queiroz Sousa
Distrito Federal - Dea Márcia da Silva M. Pereira
Espírito Santo - Carlos Urbano Gonçalves Ferreira Jr.
Goiás - Marcelo Cecílio Daher
Maranhão - Graça Viana
Mato Grosso do Sul - José Ivan de A. Aguiar
Minas Gerais - Carlos Ernesto Ferreira Starling
Pará - Helena Andrade Zeferino Brígido
Paraíba - Luciana Holmes Simões
Paraná - Alceu Fontana Pacheco Jr.
Pernambuco - Martha Maria Romeiro F. Fonseca
Piauí - Kelsen Dantas Eulálio
Rio de Janeiro - J. Samuel Kierszembaum
Rio Grande do Norte - Hênio Godeiro Lacerda
Rio Grande do Sul - Luciano Goldani
Rondônia - André Luis de Freitas Alves
Santa Catarina - Silvia Cristina C. Flores
São Paulo - Maria Luiza Moretti
Sergipe - Márcia Maria Macedo Lima
Tocantins - Hertz Ward de Oliveira
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THE BRAZILIAN JOURNAL OF INFECTIOUS DISEASES
An Official Publication of the Brazilian Society of Infectious Diseases
EDITOR
John R. David (US)
Jorge Arias (BR)
Jorge Luiz Nobre Rodrigues(BR)
Jorge Luiz Sampaio (BR)
José Wellington Oliveira Lima (BR)
Kleber Luz (BR)
Marcelo Ferreira (BR)
Marcos Antônio de Ávila Vitória (BR)
Maria Aparecida Shikanai Yasuda (BR)
Maria Rita Elmor (BR)
Mark Wainberg (CA)
Mauro Schechter (BR)
Mitermayer Galvão dos Reis (BR)
Naftale Katz (BR)
Raimundo Paraná (BR)
Reinaldo Salomão (BR)
Ricardo Diaz (BR)
Richard Guerrant (US)
Richard Locksley (US)
Richard B. Roberts (US)
Robério Dias Leite (BR)
Robert Schooley (US)
Rod Hay (GB)
Rodolfo Teixeira (BR)
Rogério de J. Pedro (BR)
Selma Maria Bezerra Jerônimo (BR)
Sérgio Cimerman (BR)
Sérgio Coutinho (BR)
Sylvia Lemos Hinrichsen (BR)
Timothy Inglis (AUS)
Warren D. Johnson, Jr. (US)
Zilton Andrade (BR)
Anastácio Q. Sousa
ASSOCIATE EDITORS
Adauto Castelo (BR)
André Villela Lomar (BR)
Antônio C. Pignatari (BR)
Carlos Brites Alves (BR)
Hélio Sader (BR)
João Silva de Mendonça (BR)
Márcio Nucci (BR)
Roberto Badaró (BR)
Roberto Focaccia (BR)
EDITORIAL BOARD
Achilea L. Bittencourt (BR)
Antônio Alci Barone (BR)
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Antônio Campos Neto (BR)
Antônio Carlos Nicodemo (BR)
Arnaldo Colombo (BR)
Caio Mendes (BR)
Celso Ramos Filho (BR)
Cláudio Sérgio Pannuti (BR)
Dirceu Grecco (BR)
Edgard Marcelino de Carvalho (BR)
Eduardo Netto (BR)
Érico Antônio Gomes de Arruda (BR)
Esper Georges Kallas (BR)
Eurico de Arruda Neto (BR)
Flávia Rossi (BR)
Guido Levi (BR)
Henry Masur (US)
Jeffrey Shaw (BR)
PRODUCTION STAFF
Luciana Bastianelli, Managing Editor
Andréia Lima, Submissions Manager
Taís Cupertino, Secretary
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The Brazilian Journal of Infectious
Diseases is an official publication of the Brazilian
Society of Infectious Diseases and is published bimonthly by Contexto - Rua Alfredo Magalhães, 04/
Barra, 40140-140, Salvador-Bahia-Brazil. The
editorial offices are at Contexto.
Editorial Office
Correspondence concerning subscriptions,
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Contributors please consult the Instructions for
Authors in this issue or visit www.bjid.com.br
Sponsors
This supplement was supported by:
• Produtos Roche Químicos e Farmacêuticos S/A
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Copyright 2007 by The Brazilian Journal of
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COVER: Liver cells infected with the hepatitis C virus.
© 2007 by The Brazilian Journal of Infectious Diseases and
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THE
BR
AZILIAN
BRAZILIAN
5
JOURNAL
OF
INFECTIOUS
Volume 11 • Supplement 1
DISEASES
October 2007
on the Management and Treatment of Hepatitis C ........... 1
Therapeutic Approach to Acute Hepatitis C ................... 49
Brazilian Society of Infectious Diseases HCV Consensus Group
Epidemiological Aspects of Hepatitis C in Brazil .............. 6
Treatment of Chronic Hepatitis C in Treatment-Naïve
Patients ........................................................................... 53
Decio Diament
Marcelo Simão Ferreira
Sexual Transmission of HCV ............................................. 8
Kleber Dias do Prado
Retreatment of Hepatitis C Patients Who Previously
Experienced Treatment Failure ......................................... 58
Hepatitis C Virus Perinatal Transmission ......................... 10
Fernando Lopes Gonçales Jr.
Umbeliana Barbosa de Oliveira
Maintenance Treatment for the Modulation of Liver
Fibrosis ........................................................................... 60
H e p a t i t i s C : Vi r o l o g i c a l A s p e c t s a n d P r a c t i c a l
Implications .................................................................... 12
Antonio Alci Barone
Rodrigo Nogueira Angerami and Fernando Lopes Gonçales Júnior
Evaldo Stanislau Affonso de Araújo and Antonio Alci Barone
Pathogenesis of Hepatitis C – HCV Consensus 2007 ...... 14
Treatment of Patients Infected with Hepatitis C Virus and
Presenting Extrahepatic Manifestations .......................... 64
Ana Tereza R. Viso
Fátima Mitiko Tengan1 and Antonio Alci Barone
Antifibrotic Therapy in Chronic Hepatitis C .................... 20
Rinaldo Focaccia Siciliano and Antonio Alci Barone
H e p a t i t i s C Tr e a t m e n t B e f o r e a n d A f t e r L i v e r
Transplant ..................................................................... 69
Laboratory Testing for Hepatitis C .................................. 21
Edson Abdala, Daniela Rosa Magalhães Gotardo, Patrícia Rodrigues
Bonazzi and Telésforo Bacchella
Neiva Sellan Lopes Gonçales and Fernando Lopes Gonçales Junior
Adverse Event Management ........................................... 74
Hepatitis C: Genotyping ................................................... 25
Aline Gonzalez Vigani
Norma de Paula Cavalheiro
Treatment Options in the Management of Thrombocytopenia
in Patients Infected with HCV .......................................... 79
Noninvasive Means of Diagnosing Liver Fibrosis in
Hepatitis C ...................................................................... 28
Eduardo Sellan Lopes Gonçales, Adriana Flávia Feltrim Angerami
and Fernando Lopes Gonçales Junior
Chronic Hepatitis C: Pathological Anatomy ..................... 32
André Cosme de Oliveira
Therapeutic Perspectives for Hepatitis C ......................... 81
Evaldo Stanislau Affonso de Araújo, Antonio Alci Barone and JeanMichel Pawlotsky
Evandro Sobroza de Mello and Venâncio Avancini Ferreira Alves
Co-Infection with Hepatitis B Virus and Hepatitis C
Virus ................................................................................ 37
Heloísa Pedrosa Mitre and João Silva de Mendonça
Co-Infection with Hepatitis C Virus and Human T Lymphocyte
Virus ................................................................................. 40
Instructions for Authors
Carlos Brites Alves
Basic Guidelines for the Treatment of HIV/HVC CoInfection ......................................................................... 42
Statement of Editorial Policy
Edgard De Bortholi
Basic Aspects of the Treatment for Hepatitis C: Mechanisms
of Action of Interferon Alpha and Ribavirin and the Bases of
Individualization .............................................................. 47
Carlos Eduardo de Melo, Evaldo Stanislau Affonso de Araújo and
Antonio Alci Barone
Checklist for Submitted Manuscripts
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6
BJID on line
The Brazilian Journal of Infectious Diseases (BJID) is
now present in a new Web Site: www.bjid.com.br. The
BJID is an official publication of The Brazilian Society
of Infectious Diseases and it is a vehicle for original
publications in this field. The BJID is published bimonthly by Contexto Publishing (Salvador/BA, Brazil)
since 1997. The aim of this site is to familiarize visitors
with BJID' contents by providing online subscriptions,
review process, submission process, abstracts, past
publications, and extend the BJID´s instructions for
authors to infectious-disease specialists worldwide. Full
instructions for authors are provided in English and
Portuguese. This new website is divided into the
following sections:
Home (A new design was established for BJID,
including spaces for advertising)
Editors (This section includes all the editorial board
of BJID)
Subscriptions (In this section, the visitor could
subscribe the BJID online)
Instructions to Authors (This section is in Portuguese
and in English, and can help the authors who want
to submit papers to BJID to follow. There are the
rules for publishing in the Journal).
Index Listing (The visitors can check the status of
the BJID in the medicus indexa round the world. In
a nearly future, we will include a service to check
the impact of each article published in BJID)
Abstracts (The visitors have free access to the
abstracts of the current year. For example: every
each issue, the abstracts will be available to be
consulted. If the visitors would like the full text, they
need to ask the permission to our office).
Publications (In this section, it will be available the
full text of last volumes 1997 to 2003 in pdf format)
Sponsors (This space is reserved to the advertising
board of the BJID and the institutions that support
the Journal. The visitors can be addressed directly to
the sponsor they want by a link)
About us (This section inform the visitors about the
production staff of the Journal, the address, phone
number, e-mail, and the contacts of the BJID)
Links (Interesting links about infectious diseases and
institutions)
This website reserved a new space to the authors
who have already submitted papers to the Journal
and/or those have papers in the submission process
in BJID with the following sections:
On-line Submission Process (The authors now could
send the papers by e-mail, following the specific rules
described in this section. Also, the authors now can
consult the review process of the submitted articles
via on-line. Immedialely after the submission of the
article, the main author will receive a login and a
password, with which the author will be able to
consult the status of the article. If other authors want
to check the status of their article via on-line, they
can also register a proper login and password.
On-line Review Process (The reviewers now can
receive the article and make all the edition process
by internet. The reviewer will receive a message
with a login an a password. With them, he could
access the entire article. After he comments in a
specific form, it will be sent to the author
automatically. The authors now can consult the
review process of the submitted articles via online. Immediately after the submission of the article,
the main author will receive a login and a password,
with which the author will be able to consult the
status of the article. If other authors want to check
the status of their article via on-line, they can also
register a proper login and password.
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BJID 2007; 11 Supplement 1 (October)
1
on the Management and Treatment of Hepatitis C
Evaldo Stanislau Affonso de Araújo, João Silva Mendonça, Antonio Alci Barone, Fernando Lopes Gonçales Junior, Marcelo
Simão Ferreira*, Roberto Focaccia, Jean-Michel Pawlotsky§* and Brazilian Society of Infectious Diseases HCV Consensus Group
Each year, and every day, the results of clinical trials and basic research provide us with a great deal of new
information regarding viral hepatitis. We on the Viral Hepatitis Committee of the Sociedade Brasileira de Infectologia
(SBI, Brazilian Society of Infectious Diseases) have been working to standardize the major issues surrounding dayto-day practice in treating patients infected with the hepatitis B or C virus (HBV or HCV). We have decided to
address, in alternate years, HBV, together with hepatitis delta (HDV), and HCV, in our annual ‘Consensus’ on
clinical management. Last year, we published the first HBV Consensus (BJID, 2007 (11):2-6). This year, we submit
our HCV Consensus,
which primarily serves to
update the 2002 and 2004
SPI Consensuses. We
distributed the principal
topics among the Committee
members, revised their
work and compiled it into a
Proceedings Supplement
(to be published together
with the BJID), which
elucidates the highlights of
the Consensus. A deeper
review was written and
referenced (it is our advice
to the reader to read the
Proceedings as well). A
meeting was then held in
Mogi das Cruzes in order
to discuss, in a very practical
and directed way, the
issues most relevant to the
Consensus, from public
policies to the most complex
therapeutic points. The
results are summarized in a
question/answer, topic/
statement format in this issue
of the BJID. The main
message of our statement was
Figure 1. HCV Consensus Group.
that we need to have the
courage to act in favor of life.
Many of us have adopted certain practices based on very new knowledge despite a lack of formal or official policies
to support such practices. Some of us have been awaiting new compounds while patients are dying of chronic liver
diseases. Unfortunately, the news from the battlefield is not so good. New compounds have been very disappointing
(low potency, viral resistance, ineffective without interferon and various side effects, some serious). It is also difficult
to incorporate new policies into everyday practice. However, strategies such as optimizing the use of pegylated
interferon/ribavirin and encouraging treatment compliance, as well as finding new ways to monitor and slow liver
disease progression, are effective and should be put into practice. Most importantly, the low-dose maintenance of
pegylated interferon seems to be very promising, and the use of interferon alpha has saved lives. That is why we
choose to expound upon what we believe to be the current standard of care and the gold standard for dealing with
this hard to treat virus, as well as with the chronic complications of HCV infection. Our position will be re-evaluated
over the next two years. Until then, we are confident that our guide will be of great value to the readers. Finally, we
would like to thank Roche and Schering Plough for the educational grants provided to the SBI. However, we must
stress that neither company attempted to influence any of the decisions made by our consensus group.
The Brazilian Society of Infectious Diseases HCV Consensus Group
Social and Collective Impact of the Decision-Making Process
Serologic triage for hepatitis C virus (HCV): When and how?
Diagnosis ‘campaigns’? Most vulnerable groups?
It is recommended that detection ‘campaigns’ be carried
out for more vulnerable groups 1, individuals in certain
#Brazilian Society Infectious Diseases HCV Consensus Group: Ana
Tereza Rodrigues Viso, Aline Vigani, André Cosme Oliveira, Carlos
Eduardo de Melo, Carlos Brites Alves, Décio Diament, Edson Abdala,
Edgard De Bortholi, Evandro Sobroza de Mello, Eduardo Sellan
Gonçalves, Fátima Mitiko Tengan, Heloísa Pedrosa Mitre*, Kleber
Dias Prado, Neiva Sellan Gonçales, Norma de Paula Cavalheiro, Rinaldo
Focaccia Siciliano, Rodrigo Nogueira Angerami*, Umbeliana Barbosa
de Oliveira, Venâncio Avancini Ferreira Alves*. § Special guest. *Members
who did not participate of the Consensus Meeting occurred in the Blue
Tree Park Hotel, Mogi das Cruzes/SP, on August 25th-26th, 2007.
occupations (health professionals, technicians whose work
potentially puts them in contact with blood), institutionalized
individuals (prisoners, under-age felons, etc.), and the family
members of patients with hepatitis C, as well as those infected
with HIV. We emphasize that such individuals should be clearly
informed that the triage diagnosis should be confirmed by a
method with more sensitivity and specificity, as well as that
confirmation of the diagnosis will not necessary imply
treatment of the infection. Age is a factor to be considered,
1
History of blood product transfusion, history of major surgery, unsafe
parenteral exposure (reusable syringes, sharing of utensils during the
use of licit or illicit injected substances, undergoing diagnostic or esthetic
therapeutic procedures involving reusable or inadequately sterilized
material, e.g., tattoo, acupuncture, piercing, manicure, dental
treatment, etc.), and sharing of utensils during the use of inhaled drugs.
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2
Consensus of Hepatitis C Carried Out by SBI
since older people are more likely to have been exposed to the
disease over the course of their lifetime.
The infected health professional: are their activities restricted
in any way?
A priori, the activity of health professionals with
hepatitis C should not be restricted. However, strict
application and educational measures are recommended
regarding the biosafety guidelines, and it should be noted
that more data are needed before a definitive
recommendation can be made.
Is hepatitis C a sexually transmitted disease (STD)?
Although hepatitis C is not conceptually an STD, sexual
transmission is possible, albeit uncommon, and the risk of
such transmission increases when the individual presents
genital lesions or HIV positivity, as well as when the
individual engages in risky sexual behavior. In such
situations, the use of condoms is recommended.
Heterosexual monogamous couples who present
discrepant serologic results can use condoms of their own
accord after being properly instructed. We would like to
issue a warning regarding the risk of transmission in the
household through the communal use of utensils
contaminated with blood.
Hepatitis C during pregnancy and childbirth: To get pregnant
or not? Cesarean or vaginal delivery? Breastfeeding?
Pregnancy is not contraindicated in women of
childbearing age infected with HCV, although
contraindications related to the period of treatment should
be respected. Regarding the type of delivery, the decision
should be made by an obstetrician. It should be noted,
however, that a high viral load of HCV can be a relevant
factor in this decision. Nevertheless, at the present, we
cannot recommend a definitive course of action.
Breastfeeding is allowed, although the nipple should be
carefully prepared, and breastfeeding should be
discontinued if fissures appear or bleeding occurs. Pregnant
women coinfected with HCV and HIV present a clearly
greater risk of perinatal HCV transmission and therefore
constitute an exception to these permissions.
Prevention
Unapparent HCV transmission in society and in the health
care environment: What should we recommend to Health
Oversight Agencies?
We recommend extremely rigorous inspection of health
and esthetic institutes, as well as continuing education
of the professionals who work in this area. We should
also emphasize the need for ongoing education of health
professionals at all levels of patient care – from basic care
to highly complex treatments.
Vaccines: Which and when?
Susceptible individuals with hepatitis C should be
vaccinated against hepatitis A and B. There is a real need to
make vaccines against hepatitis A available in the public health
care system.
The Laboratory in the Era of Individualized Treatment
Quantification of viral loads for all genotypes?
Yes, it is recommended that the viral load of all patients be
quantified.
Which quantification method should be used?
It is recommended that the method used be reproducible,
be sensitive, and present ample linearity.
‘Moments of decision’: At baseline (One measurement?
When? Several measurements?), as well as at weeks 4, 12, and
24; end-of-treatment response (ETR), sustained virologic
response (SVR)...What else?
Baseline viral load should be determined prior to but as
close as possible to the initiation of the treatment. It can be
determined only once provided that the test is performed
under ideal technical conditions. At week 4, the same test will
be qualitative in order to define the presence of rapid virologic
response (RVR), defined as the detection of no viral RNA. At
week 12, it should be quantitative, adopting the criteria of no
early virologic response (EVR) (a drop in viral load ≤ 2log10),
partial EVR (drop ≥ 2log10), and complete EVR (no viral RNA
detected). At week 24, the test will be qualitative. If viral RNA
is detected, the treatment instituted exclusively for virologic
purposes will have to be interrupted. Determination of the
ETR – emphasizing the differences in duration in different
genotypes and patients – as well as of the SVR, must be
qualitative. The SVR should be determined at 24 weeks after
the end of the treatment.
It should be noted that monitoring will be unnecessary
during the treatment in the cases in which RVR occurred and
adequate treatment compliance is maintained.
‘Week 12’ SVR?
Determination of the SVR at 12 weeks after the end of the
treatment is not currently considered a useful or valid measure.
Metabolic alterations: Homeostasis model assessment
(HOMA) and glucose tolerance test: when to order, how to
interpret
Various studies indicate the role of hepatitis C as a factor
implicated in the development of type 2 diabetes in patients at
high risk (male gender, over 40 years of age, and overweight).
In experimental models, HCV was found to induce insulin
resistance, including increased production of tumor necrosis
factor as one of the contributing factors. Insulin resistance is
also associated with the development of steatosis and
progression of liver fibrosis, principally in patients infected
with HCV genotype 1. Therefore, there seems to be an
association between insulin resistance and the characteristics
associated with patients presenting a worse response to the
hepatitis C treatment: cirrhosis, obesity, concomitant infection
with HIV (taking antiretroviral drugs), etc.
In summary: HCV promotes insulin resistance, which leads
to steatosis, fibrosis, and resistance to treatment with
interferon alpha (IFN-α).
The HOMA mathematical model [(serum levels of fasting
insulin vs. serum levels of fasting glucose)/22.5] has proven
useful in the evaluation of sensitivity to insulin; however, it
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has not been completely standardized for all clinical situations,
which is why its routine use is not recommended, except in
the situations that will be reviewed now (metabolic alterations)
and in the Proceedings.
Noninvasive monitoring of fibrosis
Value of simplicity: Thrombocytopenia as a marker of liver
cirrhosis
Thrombocytopenia in an individual with hepatitis C
indicates moderate to severe liver fibrosis. The sensitivity of
this diagnostic marker of advanced fibrosis increases when it
is used in combination with other biochemical tests, such as
determination of the aspartate aminotransferase to platelet
ratio index, calculated using the formula: aspartate
aminotransferase/alanine aminotransferase ratio + platelets.
When is it indicated?
Noninvasive monitoring of liver fibrosis should be performed
in cases of (i) contraindication or difficult access to liver biopsy,
(ii) the need for such monitoring in order to hasten the performance
of a new biopsy in patients at greater risk of progression
(immunocompromised patients) or in individuals presenting
stage F1 fibrosis, and (iii) in patients with liver cirrhosis under
treatment for modulation of fibrosis (IFN maintenance).
How to measure: Serum panels vs. Fibroscan
Sensitivity and positive predictive value are increased by
combining the methods.
Liver biopsy is dead. . . Long live liver biopsy!
Biopsy for all genotypes? And for all patients?
Yes, except for clinical contraindications, the biopsy
should be performed for all patients presenting detectable
levels of HCV RNA.
The central issue is the absolute need for the biopsy sample
to be representative of the hepatic parenchyma, since
inappropriate biopsies frequently result in understaging of
the disease. It is highly recommended that a needle biopsy be
performed (wedge biopsies produce subcapsular samples that
cannot be used in the staging of fibrosis nor in the staging of
inflammation); needles that produce very thin biopsies highly
limit architectural staging and should not be used. Trucut 14gauge needles or needles that produce samples of equivalent
diameter, measuring at least 1.5 cm long and/or 10 represented
portspaces, are recommended. If the biopsy is performed
during a surgical procedure, it should be done at the outset of
the procedure in order to avoid the artifacts that surgery can
produce in the liver tissue.
The biopsy report should include a staging system and
scoring (METAVIR or SBP, preferably). It should be noted,
however, that the pattern of inflammatory alterations (portal,
interface, and lobular components) should be described in
detail, since it has become increasingly important in predicting
the evolution of the disease as well as in the differential
diagnosis with other diseases. In addition, the principal role
of biopsy is to rule out other liver diseases – steatohepatitis
(alcoholic or nonalcoholic), for example, frequently co-exists
with hepatitis C and is known to have a significant impact on
the evolution of the disease.
3
Value of the imaging and serum level methods in the diagnosis
of liver fibrosis: who needs endoscopy?
Prior to biopsy, patients with indirect evidence of portal
hypertension (ultrasonographic signs, thrombocytopenia)
should be submitted to endoscopy of the upper digestive tract
for detection of esophageal varicose veins that would render a
liver biopsy unnecessary in the diagnosis of liver cirrhosis.
Clinical Management in Borderline Situations
Portal hypertension in clinical practice: Clinical management
of pretreatment thrombocytopenia
There is no conclusive evidence on the management of
pretreatment thrombocytopenia; however, some alternatives
can be considered and are reviewed in the Proceedings.
Liver transplant in clinical practice: post-transplant limitations.
Fibrogenesis, treatment after transplant, rejection control and
live donor
Liver disease caused by HCV corresponds to almost half
of the indications for liver transplant. Currently, in Brazil, the
distribution of the organ follows a criterion of severity, using
the model for end-stage liver disease (MELD). After the
transplant, up to 80% of the patients experience histologic
recurrence, and the pre-transplant viral load is one of the most
significant risk factors. For this reason, pre-transplant
treatment should always be considered, even in patients with
decompensated cirrhosis, provided that the treatment is given
in a specialized center and with an active transplant treatment
team. Post-transplant treatment for chronic hepatitis is
generally indicated when fibrosis is ≥ 2 or when periportal
activity is ≥ 3. Although the duration of treatment should be
at least 48 weeks, it should be individualized according to the
virologic response profile.
Treatment
Nondrug and nonspecific HCV treatment: metabolic syndrome,
nonalcoholic steatohepatitis, diabetes, obesity – evidence for
the use of diet, hypolipidemic agents, and antidiabetic
medication
It is recommended that the conditions associated with
worsening of liver fibrosis and lower SVR rate, such as obesity,
type 2 diabetes, nonalcoholic steatohepatitis, steatosis,
dyslipidemia, and metabolic syndrome, be brought under
control before antiviral treatment is instituted.
Patients with concomitant diseases; how to use IFN-α and
specific measures for drug users and patients with mental
disease. Socially marginalized individuals and prisoners.
Individuals with kidney disease in pre- and post-kidney
transplant phases. Individuals with auto-immune disease and
extrahepatic manifestations
At facilities where a multiprofessional approach is taken or at
specialized centers, both of which allow appropriate monitoring
and control of all clinical situations of the underlying disease, as
well as of those potentiated or triggered by HCV treatment, it is
recommended that HCV treatment be instituted first. It is
noteworthy that, in the case of auto-immune manifestations
(cryoglobulinemia and auto-immune hepatitis in particular),
treatment for HCV should be given – observing the premises
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4
above. Corticosteroids and other immunosuppressants can be
used concomitantly when indicated.
a high viral load (> 850,000 IU/mL). The dose of PEG-IFN-α-2b
should be 1.5 μg/kg/week.
Ribavirin: Always a full dose? And how much (0.8 g, 1 g, 11
mg/kg/day, 15 mg/kg/day)?
Ribavirin should always be given in a full dose. The
recommended dose is 15 mg/kg/day or 1 g for patients who
weigh < 75 kg. The minimum dose to be considered in
adjustments is 11 mg/kg/day.
Short, long, standard treatment ... Always individualize?
In patients presenting a RVR, no comorbidities (severe
steatosis or cirrhosis), and a low viral load (< 250,000 IU/
mL for genotype 1), the duration of the treatment can be
reduced: to 12 weeks for genotypes 2 and 3; and to 24
weeks for genotype 1. Patients with a high viral load and
complete EVR should be treated for the standard duration,
and patients presenting a slow response (no RVR, partial
EVR and negative at week 24) should be treated for an
additional 24 weeks.
Common adverse effects: how to deal with anemia, neutropenia
and thrombocytopenia before the treatment, during the
treatment, and triggered by the treatment? What are the basal
limits for patients with and without cirrhosis to be treated?
How can the effects be reversed?
The minimum criteria for treatment should be hemoglobin
≥ 10 g/dL, neutrophils > 1500/mm3 and platelets > 60,000/mm3.
Patients presenting a drop in hemoglobin to < 10 g/dL or a
drop ≥ 3.5 g/dL with clinical manifestations should be given
Erythropoietin in a dose of 40,000 IU/week until the end of the
treatment or until anemia is under control. Patients in whom
neutrophils drop to < 750/mm3 should receive filgrastim in a
dose of 300 µg one to three times a week until neutropenia is
under control. In patients presenting a drop in platelets to <
25.000/mm3, the treatment should be discontinued. Adjustment
regimens of the dose of IFN with intermediate values are
presented in the Proceedings.
When to treat: the patient, the doctor, the system ...
The patient wants to be treated. How can we deal with this?
There is no definite position on this situation. We
emphasize the need to give clear and exhaustive information
regarding the indications for treatment and the factors
associated with the progression of the disease.
Is early indication of treatment worthwhile? In which cases?
Treatment can be considered for individuals with level 1
structural lesion (F1 METAVIR), who present incomplete septa,
A3 activity (METAVIR), who are under 60 years of age, and
present co-factors associated with risk of progression (obesity,
nonalcoholic steatohepatitis, etc.).
Use of IFN and ribavirin
The patient, the virus, and the medication: Relevant factors
in predicting response before and during the treatment
There has been no relevant alteration regarding the
elements described. Please see the concepts listed below
regarding individualization based on EVR.
Is conventional treatment with IFN-α dead? If not, when
should we use it?
It is the understanding of the Brazilian Society of Infectious
Diseases that treatment with conventional IFN-α is no longer
justifiable and should not be performed.
Dose of IFN, the ‘burden’ of the ‘weight’
The dose of pegylated IFN-α-2a (PEG-IFN-α-2a) should
be 270 μg/week for patients weighing > 85 kg and presenting
The fear of suspending treatment: How should we deal with
that decision?
We emphasize that, if the objective of the treatment is
virologic, the absence of EVR and viral detection at week 24
necessarily imply the interruption of the treatment.
Maintenance treatment with IFN: When and how? Is
‘watchful waiting’ still a valid concept?
Maintenance treatment with PEG-IFN-α is considered for
the following patients: those with structural lesion > F3 and/
or signs of portal hypertension (esophageal varicose veins,
splenomegaly, dilated blood vessels, etc.) and/or platelet
counts < 110,000/mm3 who are categorized as Child-Pugh class
A or B, with no history of severe or potentially uncontrollable
decompensation; those without hepatocellular carcinoma;
relapsers; and partial responders or nonresponders to the
combination of PEG-IFN-α and ribavirin (or to IFN only when
ribavirin is contraindicated) administered with adequated
compliance and for a minimum of 12 weeks. Maintenance
treatment with PEG-IFN-α should also be considered for
patients for whom the full dose treatment is contraindicated.
Child-Pugh class C patients on the transplant waiting list
should be treated in specialized centers. Proposed regimen2:
PEG-IFN-α-2b, 0.5-1.0 μg/kg/week subcutaneously (sc); PEGIFN-α-2a, 90 μg/week (sc). Duration2: Minimum of 24 months,
indefinite, or even until there is an antiviral treatment that is
proven to be safe and efficient. Monitoring the occurrence of
complications (hepatocellular carcinoma, gastrointestinal
bleeding, encephalopathy, etc.)
Monotherapy with ribavirin, although previously
described, cannot be recommended at the moment, due to
insufficient evidence.
Impact of reduction in the dose of IFN and/or ribavirin at
various moments of the treatment
Every effort should be made to always maintain full
treatment.
Positive and negative predictive values in clinical practice:
patients mono- and co-infected with HIV
Positive predictivity data in the presence of RVR and
negative predictivity data in the absence of EVR are equally
2
Dose schedule and duration were suggested based on preliminary data
and should be re-evaluated taking into account the results of ongoing
studies.
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valid for HCV mono-infected patients and patients co-infected
with HCV and HIV.
Retreatment: When and how?
Retreatment will always be considered when the previous
treatment was considered suboptimal for the current concepts
(poor compliance, insufficient doses, inappropriate interruptions,
inadequate management of adverse events, uncontrolled
comorbidities, etc.) and when there is recurrence. The treatment
in true nonresponders to PEG-IFN and ribavirin presents such
a low chance of success that it cannot be routinely
recommended.
Due to the fact that relapsers/nonresponders to initial
treatment constitute a quite heterogeneous group, it is
necessary to qualify and carefully select the patients that should
be retreated. Various factors that might have influenced the
nonresponse should be modified before the initiation of or even
during the new therapeutic cycle. Currently, patients considered
less likely to respond to retreatment are those who are true
nonresponders, those who are of the Black race, those infected
with genotype 1, those with high viral loads, those with
advanced liver disease, and those who present intercurrent
conditions (obesity, etc.)
Patients previously treated with IFN as monotherapy or
with the combination of IFN and ribavirin stand a greater chance
of presenting an SVR than do nonresponders to the
combination of PEG-IFN and ribavirin. Those who experienced
recurrence during the treatment (breakthrough) or after the
treatment fare better than do true nonresponders. Patients who
are noncompliant with previous treatment, as well as those
who required reduced doses of IFN or ribavirin due to cytopenia
or other adverse effects, usually respond better to retreatment
than do those who received full doses. Of course, the factors
responsible for noncompliance should be eliminated, and
reductions in medication doses should be properly approached.
In nonresponders, it is recommended that erythropoietin and
filgrastim be started sooner, and further dose reductions should
be avoided at all costs. Users of drugs or alcohol who, due to
their addiction, did not adequately comply with all the phases
of the previous treatment, can more adequately respond to
retreatment provided that these co-factors are nullified. This
also applies to patients who did not receive adequate social
or cultural support. Patients who suffer from obesity, insulin
resistance, dyslipidemia, steatosis, anemia, or liver diseases
(e.g., hemochromatosis) should be retreated, preferably
after the proper diagnosis and treatment of these
concomitant conditions. Doses of PEG-IFN should be the
same as those used for treatment-naïve patients. We believe
that, in retreatment regimens, the doses of ribavirin should
be as high as possible. Due to the paucity of studies with
large patient samples, we recommend that the duration of
retreatment be 48 weeks for all genotypes. At the moment,
there are no consolidated data in the literature to support
using higher doses of PEG-IFN or ribavirin, using induction
doses, or extending the treatment time to more than 48
weeks in retreatment cases. Regarding the week 12 rule,
there is strong evidence that patients who do not present
negativity for HCV RNA by this time will have very little
chance of presenting an SVR, and their treatment should be
interrupted.
5
IFN: Backbone of the current treatment. And the future?
Perspectives and frustrations
Preliminary results of developing therapies show that IFNα will still be the active principle of the treatment for many
years. For this reason, we have adopted several of the practices
that are presented here.
Co-infections
Antiretroviral therapy 2007 and HCV treatment: relevant
interactions
Although ribavirin can reduce the concentration of some
antiretroviral nucleoside analog reverse transcriptase inhibitors
(NARTIs), there seem to be no clinical consequences.
The adverse effects of NARTIs have been associated with
HCV co-infection, female gender, obesity and prolonged exposure
to these drugs. The principal combinations with less hepatic
repercussion among NARTIs are as follows: lamivudine + abacavir;
lamivudine + zidovudine; lamivudine + abacavir + zidovudine, and
emtricitabine + tenofovir. However, current studies indicate that
ribavirin interferes with the effect of abacavir. Therefore, ribavirin
should be used with caution in patients receiving the highly active
antiretroviral therapy regimen and being treated for HCV.
Greater toxicity is principally caused by concomitant use of
didanosine and ribavirin.
CD4 counts: Minimum limits for treatment and decision-making in
patients with reconstituted immunity or immunocompetent patients
Precise indication of therapy in HIV/HCV co-infected
patients can be made in patients with CD4 counts > 350 cells/
mm3.. In patients with CD4 counts between 200 and 350 cells/
mm3, the decision to treat HCV infection should take into
account other factors, such as the duration of HCV infection,
the severity of the liver disease, the level of HIV suppression
and classical predictors of treatment response to HCV, such as
genotype and viral load.
The SVR can be predicted when HCV RNA is undetectable
in serum by week 4 of treatment. However, a < 2log10 IU/mL
reduction in viral load by week 12 and/or viremia detected at
week 24 predict a lack of virologic response, and discontinuation
of the treatment is indicated.
Ongoing studies are evaluating 72-week maintenance therapy
in co-infected patients and week-24 nonresponders. This might
be the truly appropriate treatment duration for HIV-positive
patients, even with longer time and smaller fractionated doses.
Is the immunocompetent HIV/HCV co-infected patient ‘monoinfected’?
Yes, the evolution is quite similar.
Multiple viral infections: Who should we treat first and how?
Care should be given to the dominant virus between HBV
and HCV. When treating the dominant virus, the other might
become active. We need to quantify the HBV DNA in order to
manage this situation.
The HTLV apparently modulates the host immune
response, a fact that should be considered and which is
detailed in the Proceedings.
There is no consensus regarding the correct course of
action in multiple co-infections.
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6
Epidemiological Aspects of Hepatitis C in Brazil
Decio Diament
Emílio Ribas Institute of Infectious Diseases; São Paulo, SP, Brazil
The true dimension of the epidemiological situation of
chronic hepatitis C in Brazil remains unknown. According to
data from the National Ministry of Health, 52,489 cases were
diagnosed in the period from 1994 to 2005. However, the data
regarding the final year of this series are incomplete. There is
a trend toward an increase in the number of diagnosed cases
during this period, since diagnostic techniques have become
more widely available, both in public and private health care
facilities (Figure 1).
Figure 1. Number of diagnosed cases of hepatitis C reported
by the National Ministry of Health, from 1994 to 2005, totaling
52,489 cases.
Most diagnosed cases occur in the Southeast, followed
by the South, Central-west, Northeast and North (Figure 2).
Figure 2. Distribution of cases by macroregion (National
Ministry of Health).
The Brazilian Journal of Infectious Diseases
2007;11 (5) Suppl. 1:6-7.
© 2007 by The Brazilian Journal of Infectious Diseases and Contexto
Publishing. All rights reserved.
This distribution reflects the greater availability of
resources as well as the highly populated areas in the
southeastern and southern regions of the country. However,
serological surveys carried out in the Central-west and
Northeast, as well as in the Federal District, showed prevalence
rates similar to those of a previous survey carried out in the
city of São Paulo (Figure 3).
Figure 3. Prevalence of hepatitis C in serological surveys in
several regions of the country (Galizzi Filho J, personal
communication, for the data of the surveys on the Centralwest, Federal District and Northeast; Focaccia R., personal
communication, for the data on São Paulo).
Data obtained in these serological surveys indicate
prevalence rates from 0.28 to 2.61%. According to the 2000
census taken by the Brazilian Institute of Geography and
Statistics, Brazil has a population of approximately 170 million.
It has been shown that, of those testing positive for infection
with the hepatitis C virus (HCV), approximately 80% have the
chronic form of hepatitis C. Based on these data, we can
estimate that there are 400,000 to 3,800,000 cases of chronic
hepatitis C in Brazil. Comparing these numbers with the data
from the National Ministry of Health, we can conclude that
there are a great number of undiagnosed cases of the disease.
Since there has been a trend toward an increase in the number
of diagnosed cases, there will be a significant increase in the
demand for diagnosis and treatment of chronic hepatitis C in
the future.
Another interesting fact is that only 17,204 (33%) of the
52,493 cases of chronic hepatitis reported to the Center for
Epidemiological Surveillance of São Paulo, from 1998 to 2006,
were confirmed as being hepatitis C. Another 10,690 cases
(20%) were classified as inconclusive for hepatitis C, 2099
(4%) were excluded, and 416 (0.79%) were cases of HBV/HCV
co-infection. The remaining cases were confirmed as hepatitis
B, HBV/HDV co-infection, or cases under investigation.
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Hepatitis C in Brazil
Of the 17,204 confirmed cases, the principal form of
contamination was parenteral (use of injected illicit drugs, as
well as blood and blood product transfusions), followed by
sexual transmission. Work-related accidents accounted for
few of the cases (Figure 4).
Figure 4. Distribution of cases of chronic hepatitis C by form
of transmission in 17,204 individuals in the state of São Paulo,
from 1998 to 2006 (Source: Center for Epidemiological
Surveillance, São Paulo).
Transmission was classified as unknown in 40% of the
reported and confirmed cases, as can be seen in Figure 4. This
suggests that the investigation of the form of transmission
should be improved, and that items such as administration of
medication using nondisposable syringes, acupuncture,
tattoos, and piercings should be included in the
epidemiological investigation. Another unusual fact is sexual
transmission in 10% of the cases, well above the expected,
which is less than 1%.
Another aspect to be considered is the occurrence of
hepatitis C in groups of differentiated risk, that is, unusual
7
cases such as those described in Figure 4. The first group
would be that of the manicurists, since it is supposed that the
community use of instruments by these professionals would
expose them to a higher risk of contamination. There is no
evidence that this could happen, and a recent serological
survey conducted in the city of São Paulo in beauty salons
located at shopping malls in various neighborhoods did not
demonstrate prevalence rates different from those of the
population in general (Oliveira, ACDS – personal
communication).
Another group is that of the dentists. In Brazil, localized
serological surveys with small samples of patients have
shown a low prevalence rate, ranging from 0.4 to 0.7%.
However, a study on the level of knowledge of these
professionals about the disease has shown it is very low,
demonstrating the need of greater emphasis on training and
continuing education, aiming at making them capable of
properly protecting themselves, as well as of preventing
transmission to patients and perhaps even help the
diagnosis, since several extrahepatic manifestations appear
in the mouth cavity, such as oral liquen planus, erythema
nodosum, etc.
References
1. Bellíssimo-Rodrigues W.T., Machado A.A., Bellíssimo-Rodrigues
F., et al. Prevalence of hepatitis B and C among Brazilian dentists.
Infect Control Hosp Epidemiol 2006;27:887-8.
2. h t t p : / / p o r t a l . s a u d e . g o v . b r / p o r t a l / s a u d e /
visualizar_texto.cfm?idtxt=25340. Acessado em 24/08/2007.
3. http://www.cve.saude.sp.gov.br/htm/hepa_home.html. Acessado em
24/08/2007.
4. Leão J.C., Teo C.G., Porter S.R. HCV infection: aspects of
epidemiology and transmission relevant to oral health care
workers. Int J Oral Maxillofac Surg 2006;35(4):295-300.
5. Takahama A.J., Tatsch F., Tannus G., Lopes M.A. Hepatitis C:
incidence and knowledge among Brazilian dentists. Community
Dent Health 2005;22(3):184-7.
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8
Sexual Transmission of HCV
Kleber Dias do Prado
Although there is evidence that sexual transmission of
HCV occurs, this form of transmission is of secondary
epidemiological importance when compared to percutaneous
forms of transmission [1]. Among the evidence that supports
the possibility of sexual transmission of HCV we can list the
following:
1- Case reports of acute hepatitis C with anti-HCV
seroconversion in sexual partners of individuals infected by
HCV, excluding mechanisms of nonsexual transmission and
with high genomic homology among viral strains infecting
sexual partners [2-4].
2- Detection of HCV RNA in semen, vaginal secretion and
cervical secretion, despite low titles in most cases [5-8].
3- Data from the Centers for Disease Control and Prevention
demonstrating that, from 1995 to 2000, 18% of the cases of
acute HCV infection in the United States occurred in patients
reporting sexual contact with an individual infected with HCV
in the preceding 6 months or multiple sexual contacts as the
only risk factors for acquiring the infection [1].
The risk of acquiring HCV through sexual contact differs
among subgroups of individuals: We can distinguish two main
risk subgroups:
1. Individuals who have multiple sexual partners or who
engage in sexual practices that might lead to mucosal trauma:
sex professionals; men who have sex with men (MSM); and
patients treated in clinics specializing in the treatment of
sexually transmitted diseases (STDs).
2. Stable monogamous heterosexual sexual partners of
individuals chronically infected with HCV.
In general, rates of anti-HCV incidence and prevalence are
higher in the first subgroup. This may be due to differences in
sexual practices among the groups, but also to nonsexual
factors (sharing personal objects, tattoos, use of illicit drugs,
etc.) [1]. Seroprevalence studies in the United States
demonstrated median positive anti-HCV rates in 6% of women
who were sex professionals, as well as in 4% of MSM, 4% of
clients of STD clinics and 4% of participants in HIV
surveillance studies. Studies conducted in other parts of the
world have obtained similar results [9-15]. The following risk
factors were identified: having had a high number of recent
and lifetime sex partners; engaging in unsafe sexual practices;
being infected with HIV; and having an STD. This indicates
that sexual activity in general is a risk factor for HCV
2007;11 (5) Suppl. 1:8-9.
transmission [1].
However it is essential to mention that the results of some
studies contradict these findings. Studying the prevalence
and incidence of positivity for anti-HCV antibodies in a cohort
of 1085 HIV-positive Canadian MSM, Alary et al. found values
of 2.9% and 0.038/100 individuals/year, respectively, both
significantly associated with the use of injection drugs. The
authors considered sexual transmission of HCV to be rare in
this group [16]. In a study conducted in Thailand, Taketa et al.
assessed the prevalence of anti-HCV in injection drug users,
sex professionals and individuals with STDs. The prevalence
was 85%, 2% and 0%, respectively, with a very low or null
transmission rate in the last two groups [17]. Marincovich et
al. prospectively studied a group of 171 couples discordant
for HIV and HCV. The index cases were 152 men and 19 women,
whereas the spouses were 152 women and 19 men. Fortythree per cent had engaged in unprotected vaginal and/or
anal sex, 15% always used a condom but reported incidents in
which the condom broke or slipped off during sexual contact,
and 22% had performed unprotected orogenital sex. There
was only one case of HIV seroconversion and no cases of
HCV seroconversion during the follow-up of 529 individuals/
year. There were 31 cases of pregnancy, 2 of them in women
infected with HCV. This study suggests that the rate of HCV
transmission is low or null among heterosexuals, even when
the partner is infected with HIV [18].
For the second subgroup, the best studies are those
excluding percutaneous factors of infection and evaluating
genotypes and genomic sequence of viral strains in anti-HCV
concordant couples. In those studies, the prevalence of HCV
was estimated at 2.8-11% in the Asian Southeast, 0-6.3% in
Northern Europe and 2.7% in the United States [1].
In one of the first studies to use genotyping and analysis
of the sequence of nucleotides of the hypervariable E2 region,
Zylberberg et al., studying 24 anti-HCV concordant couples,
reduced to 3 couples the possible cases of sexual transmission
of HCV. Nevertheless, nonsexual factors could not be ruled
out and might have contributed to HCV transmission between
couples [19]. In Iran, Hajiani et al. studied the HCV transmission
rate for home contacts with no percutaneous risk factors. The
rates found were 1.33% for the contacts and 1% for the controls
(p>0.06). Only 2 of 59 spouses presented evidence of infection
(3.39%). The authors conclude that intrafamily transmission
is possible, although not common [20].
In a recent study, McMahon et al. determined that the
transmission of HCV in 265 heterosexual couples using drugs
in New York City was associated with the use of injection
drugs by the couple, although not with the pattern of sexual
activity [21]. Along the same lines, Boonyarad et al., studying
160 spouses infected with chronic hepatitis C (106 women
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Sexual Transmission of HCV
and 54 men, all monogamous, stable heterosexual couples),
verified that only 3 individuals (1.88%) tested positive for
anti-HCV antibodies and HCV RNA after a mean period of 23
± 5 years of unprotected sexual exposure. Nevertheless, in
those 3 individuals, genotyping and sequence analysis did
not clearly identify the same viral strains that infected their
respective partners. The authors concluded that sexual
transmission of HCV is rare [22].
Finally, Vandelli et al. conducted a large prospective study
in which 895 monogamous and stable heterosexual couples
were evaluated for 10 years [23]. Among the spouses, the
authors identified 3 cases of seroconversion, none of which
were attributed to sexual contact with the partner: in one case,
the genotypes were different; in the other two, there were
discrepancies in the sequence and phylogenetic analysis).
Therefore, the authors conclude that the rate of sexual
transmission of HCV is very low or even null in these patients.
Therefore, it seems unnecessary to recommend condom use
in this population. These couples did not practice anal sex,
neither sex during menstruation nor used condoms [23].
The risk of sexual transmission of HCV ranges from 00.6%/year for heterosexual couples in monogamous, stable
relationships to 1%/year to individuals with several sex
partners [1]. Therefore, the formal, systematic recommendation
of condom use is only necessary for the latter group. Condom
use is also justified in HCV-positive individuals presenting
concomitant STDs, having sex during menstruation or
engaging in sexual practices that can traumatize the mucosal
surfaces (anal sex, fisting, etc.) In conclusion, the sharing of
objects potentially contaminated with blood, such as razor
blades, scissors, nail clippers, cuticle trimmers and tooth
brushes, is not recommended [1].
References
1. Terrault N.A. Sexual activity as a risk factor for hepatitis C.
Hepatology 2002;36:S99-105.
2. Halfon P., Riflet H., Renou C., et al. Molecular evidence of maleto-female sexual transmission of hepatitis C virus after vaginal
and anal intercourse. J Clin Microbiol 2001;39:1204-6.
3. Nakayama H., Sugai Y., Ikeya S., et al. Molecular investigation of
interspousal transmission of hepatitis C virus in two Japanese
patients Who acquired acute hepatitis C after 40 or 42 years of
marriage. J Med Virol 2005;75(2):258-66.
4. Lai K.W., Young K.C., Cheng P.N., et al. Interspousal transmission
of hepatitis C virus: application of comparing the variability of
HVR1
nucleotide
region.
Hepatogastroenterology
2004;51(57):791-5.
5. Leruez-Ville M., Kunstmann J.M., De Almeida M., et al. Detection
of hepatitis C virus in the semen of infected men. Lancet
2000;356:42-3.
6. Manavi M., Watkins-Riedel T., Kucera E., et al. Evidence of
hepatitis C virus in cervical smears. J Infect 1999;38:60-1.
7. Pekler V.A., Robbins W.A., Nyamathi A., et al. Use of versant
TMA and bDNA 3.0 assays to detect and quantify hepatitis C
virus in semen. J Clin Lab Anal 2003;17(6):264-70.
9
8. Bélec L., Legoff J., Si-Mohamed A., et al. Cell-associated, nonreplicating strand (+) hepatitis C virus-RNA shedding in
cervicovaginal secretions from chronically HCV-infected
women. J Clin Virol 2003;27(3):247-51.
9. Van de Laar T.J., Van der Bij A.K., Prins M., et al. Increase in HCV
incidence among men who have sex with men in Amsterdam
most likely caused by sexual transmission. J Infect Dis
2007;196(2):230-8.
10. Danta M., Brown D., Bhagani S., et al. Recent epidemic of acute
hepatitis C virus in HIV-positive men who have sex with
men linked to high-risk sexual behaviours. AIDS
2007;21(8):983-91.
11. Götz H.M., Van Doornum G., Niesters H.G., et al. A cluster of acute
hepatitis C virus infection among men who have sex with men:
results from contact tracing and public health implications.
AIDS 2005;19(9):969-74.
12. D’Oliveira A. Jr., Voirin N., Allard R., et al. Prevalence and sexual
risk of hepatitis C virus infection when human immunodeficiency
virus was acquired through sexual intercourse among patients of
the Lyon University Hospitals, France, 1992-2002. J Viral Hepat
2005;12(3):330-2.
13. De los Angeles Pando M., Biglione M.M., Toscano M.F., et al.
Human immunodeficiency virus type 1 and other viral coinfections among young heterosexual men and women in
Argentina. Am J Trop Med Hyg 2004;71(2):153-9.
14. Marx M.A., Murugavel K.G., Tarwater P.M., et al. Association of
hepatitis C virus infection with sexual exposure in southern
India. Clin Infect Dis 2003;37(4):514-20.
15. Russi J.C., Serra M., Viñoles J., et al. Sexual transmission of hepatitis
B virus, hepatitis C virus, and human immunodeficiency virus
type 1 infections among male transvestite commercial sex
workers in Montevideo, Uruguay. Am J Trop Med Hyg
2003;68(6):716-20.
16. Alary M., Joly J.R., Vincelette J., et al. Lack of evidence of sexual
transmission of hepatitis C virus in a prospective cohort study
of men who have sex with men. Am J Public Health
2005;95(3):502-5.
17. Taketa K., Ikeda S., Suganuma M., et al. Differential
seroprevalences of hepatitis C virus, hepatitis B virus and human
immunodeficiency virus among intravenous drug users,
commercial sex workers and patients with sexually transmitted
diseases in Chiang Mai, Thailand. Hepatol Res 2003;27(1):6-12.
18. Marincovich B., Castilla J., Del Romero J., et al. Absence of hepatitis
C virus transmission in a prospective cohort of heterosexual
serodiscordant couples. Sex Transm Infect 2003;79(2):160-2.
19. Zylberberg H., Thiers V., Lagorce D., et al. Epidemiological and
virological analysis of couples infected with hepatitis C virus.
Gut 1999;45(1):112-6.
20. Hajiani E., Masjedizadeh R., Hashemi J., et al. Hepatitis c virus
transmission and its risk factors within families of patients
infected with hepatitis C virus in southern Iran: Khuzestan.
World J Gastroenterol 2006;12(43):7025-8.
21. McMahon J.M., Pouget E.R., Tortu S. Individual and couple-level
risk factors for hepatitis C infection among heterosexual drug
users: a multilevel dyadic analysis. J Infect Dis
2007;195(11):1556-9.
22. Boonyarad V., Chutaputti A., Choeichareon S., et al. Interspousal
transmission of hepatitis C in Thailand. J Gastroenterol
2003;38(11):1053-9.
23. Vandelli C., Renzo F., Romanò L., et al. Lack of evidence of sexual
transmission of hepatitis C among monogamous couples: results
of a 10-year prospective follow-up study. Am J Gastroenterol
2004;99(5):855-9.
www.bjid.com.br
10
Hepatitis C Virus Perinatal Transmission
Umbeliana Barbosa de Oliveira
The hepatitis C virus (HCV) is the most frequent cause of
chronic hepatic disease. Its principal route of transmission is
exposure to contaminated blood. Perinatal transmission is one
of the less common modes of infection with HCV.
Incidence
Vertical transmission of HCV has been reported in
numerous studies. However, the estimated rate of vertical
transmission varies considerably due to several factors:
• Study methodology;
• Selection of maternal population;
• Risks involved in the transmission, such as coinfection with HIV and high levels of HCV RNA.
A review of the literature carried out in 1998 identified 976
children from 28 studies with follow-up periods that were
sufficiently long to estimate the transmission rate. In those
studies, the vertical transmission rate was below 10% in cases
involving HIV-negative mothers. The risk of vertical
transmission increased significantly in cases involving HIVpositive mothers. It is believed that, in these cases, the mother
tends to present higher levels of HCV viremia, increasing the
risk of transmission.
In a more recent review (published in 2001), the authors
searched through studies published in the 1992-2000 period
and identified 77 studies. They found clinical and demographic
variables that influenced the transmission rate. They
concluded that co-infection with HIV is the most significant
factor associated with the risk of vertical HCV transmission.
Findings regarding other possible risk factors, such as HCV
genotype, type of delivery, and breastfeeding, have been
inconclusive.
Risk Factors
As previously mentioned, the principal risk factor for vertical
transmission of HCV is concomitant infection with HIV.
Viremia Level
Some studies have shown that viral load can be an
important determinant factor for vertical transmission of HCV.
It has been demonstrated that women with viral loads lower
than 1 × 105 copies per mL have a lower risk of vertical
transmission, whether co-infected with HIV or not. One study
that compared viral loads among HCV RNA-positive mothers
whose children were infected with HCV or not showed that, in
cases of probable vertical transmission, the maternal viral load
was ten times higher than in those cases in which transmission
did not occur.
However, there are also inconsistent data in two large
studies that found no significant differences between the HCV
RNA levels of the mothers who transmitted HCV and those of
mothers who did not.
2007;11 (5) Suppl. 1:10-11.
Co-Infection with HIV
Numerous studies have shown that the HCV vertical
transmission rate is higher among HIV-positive women that
among those who are HIV-negative (19% vs. 4%). This is in
part explained by the fact that HIV-positive women present
higher viral loads of HCV.
One study showed that the average level of HCV RNA in
women co-infected with HIV was ten times higher than in
women mono-infected with HCV. This shows that the increase
in the co-infection risk can be nullified after adjusting the risk
for the viral load, and antiretroviral therapy for HIV can reduce
the risk of HCV transmission.
One study that compared HIV-positive women and HIVnegative women with similar viral loads of HCV showed that the
risk of HCV transmission was similar in both groups. In that study,
all HIV-positive women were receiving antiretroviral therapy.
Another study showed that children infected with HIV
are at a higher risk of being infected with HCV than are those
who are HIV-negative (17.1% vs. 5.4%). The explanation for
these data remains unclear.
Genotype
There is no evidence that HCV genotype influences
vertical transmission. The data collected to date do not allow
us to establish a relationship between HCV genotype and
vertical transmission risk.
Breastfeeding
It is possible to detect HCV RNA in breast milk and
colostrum. However, HCV transmission through breastfeeding
has not been documented. A likely explanation would be that
HCV is inactivated by the effect of gastric acidity and that the
HCV RNA levels in breast milk are very low.
Therefore, breastfeeding does not increase the risk that
HCV will be transmitted from an infected mother to her child.
The American College of Obstetricians and Gynecologists, as
well as the American Academy of Pediatrics, allows
breastfeeding by mothers who are infected with HCV.
It is important to emphasize that breastfeeding is not
recommended in case of nipple fissure, due to the
possibility of bleeding.
Type of Delivery
The impact that the type of delivery has on perinatal
transmission of HCV is completely unknown. Vaginal delivery
has been associated with an increase in the transmission risk.
In a study carried out by Lin et al. in 1994, 70 pregnant women
were evaluated. The vertical transmission rate among children
who were born by vaginal delivery was 32%, compared to 6%
in cases of cesarean section. However, the high transmission
rate in the Lin et al. study was associated with the fact that a
large proportion of the women evaluated (76%) were coinfected with HIV and HCV. Therefore, cesarean section is
associated with a decrease in the HCV transmission risk in co-
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Hepatitis C Virus Perinatal Transmission
infected women. The higher risk of transmission in vaginal
delivery might be associated with exposure of the newborn to
HCV-contaminated blood during its passage through the
vagina. However, the exposure to blood can sometimes be
greater in cesarean section.
Many studies are needed in order to estimate the risk of
perinatal transmission of HCV. Currently, cesarean sections
are not recommended for pregnant women infected with HCV.
Other risk factors associated with delivery are rupture of the
placental membrane six hours or more before delivery and
invasive procedures such as internal monitoring of the fetus
(Blood 2000; 96:2045).
Clinical Manifestations
Newborns infected with HCV are generally asymptomatic.
In these cases, a significant proportion presents normal
alanine aminotransferase (ALT) levels.
One study monitored 104 children with vertical
transmission upon birth for approximately 49 months (J Infect
Dis 2000;181:419). Although HCV RNA levels were positive
for HCV in 90% of these children, the clinical manifestations
were rare. None of them presented hepatic insufficiency. The
majority presented persistent or transitory increase in ALT
levels. Levels of ALT were, in general, normal or slightly
increased at birth, evolving to an increase in the fourth to
sixth months of age, remaining high for two years, and declining
significantly thereafter.
In that study, 20 children were submitted to hepatic biopsy.
In all cases, evidence of chronic hepatitis was found. The
degree of inflammation varied from mild to moderate. In three
cases some level of fibrosis was found.
Other studies suggest that children infected with verticallytransmitted HCV are generally asymptomatic. However, such
transmission is often accompanied by an increase in ALT
levels during the first 6 to 12 months of life.
One large study monitored 266 perinatally HCV-infected
children for an average of 4.2 years. In that study, approximately
20% of the children presented spontaneous clearance of HCV,
and 80% evolved to chronic infection.
Clearance of HCV is defined as a loss in the polymerase
chain reaction positivity for HCV RNA in those children who
maintained detectable HCV RNA in the first year of life. Most
of those children were asymptomatic.
Further studies involving long-term follow-up evaluation
are needed in order to determine what proportion of children
with chronic hepatitis C caused by vertical transmission will
develop hepatic insufficiency and hepatocarcinoma.
Diagnosis
The initial diagnosis of infection with HCV in adults is
made through anti-HCV antibody testing. However, this test
presents limitations when used in children.
This is primarily due to the passage of IgG from the mother
to the child through the placental barrier. Therefore, the
presence of anti-HCV in the serum of the child does not
necessarily indicate HCV infection. Clearance of the anti-HCV
passively acquired from the mother can take more than 12
11
months, although it occurs within 12 months in 95% of the
cases.
Second, the presence of maternal anti-HCV does not
necessarily mean that the mother is chronically infected with
HCV.
A diagnosis of vertical transmission is made by identifying
HCV RNA in the mother and child.
It is important to emphasize that the HCV RNA test can
also present false-positive or false-negative results. A falsepositive result can occur as a result of contamination of positive
samples. A false-negative result occurs due to a loss of RNA
during sample storage or to an error in RNA extraction.
A consensus from the National Institutes of Health
recommends that children born to mothers testing positive
for HCV should be submitted to the HCV RNA test on two
occasions: between 2 and 6 months of life and after 15
months of life (together with anti-HCV tests on the latter
occasion).
Conclusions
The incidence of vertical transmission by HCV is
approximately 2% to 5%. The risk is higher in the following
situations: co-infection with HIV; and high maternal viral load
of HCV.
There are still no effective interventions to reduce the risk
of HCV transmission from the mother to the child. As yet,
HCV testing is not recommended for pregnant women.
References
1. Manzini P., Saracco G., Cerchier A., et al. Human
immunodeficiency virus infection as risk factor for mother-tochild hepatitis C virus transmission; persistence of anti-hepatitis
C virus in children is associated with the mother’s anti-hepatitis
C virus immunoblotting pattern. Hepatology 1995;21:328.
2. Thomas S.L., Newell M.L., Peckham C.S., et al. A review of
hepatitis C virus (HCV) vertical transmission: risks of
transmission to infants born to mothers with and without HCV
viraemia or human immunodeficiency virus infection. Int J
Epidemiol 1998;27:108.
3. Yeung L.T., King S.M., Roberts E.A. Mother-to-infant transmission
of hepatitis C virus. Hepatology 2001;34:223.
4. Terrault N. Epidemiological evidence for perinatal transmission
of hepatitis C virus. Viral Hepatitis Reviews 1998;4:245.
5. Lin H.H., Kao J.H., Hsu H.Y., et al. Possible role of high-titer
maternal viremia in perinatal transmission of hepatitis C virus.
J Infect Dis 1994;169:638.
6. Kumar R.M., Shahul S. Role of breast-feeding in transmission of
hepatitis C virus to infants of HCV-infected mothers. J Hepatol
1998;29:191.
7. Azzari C., Resti M., Moriondo M., et al. Vertical transmission of
HCV is related to maternal peripheral blood mononuclear cell
infection. Blood 2000;96:2045.
8. Tovo P.A., Pembrey L.J., Newell M.L. Persistence Rate and
Progression of Vertically Acquired Hepatitis C Infection. J Infect
Dis 2000;181:419.
9. Vogt M., Lang T., Frosner G., et al. Prevalence and clinical outcome
of hepatitis C infection in children who underwent cardiac surgery
before the implementation of blood-donor screening. N Engl J
Med 1999;341:866.
10. Three broad modalities in the natural history of vertically acquired
hepatitis C virus infection. Clin Infect Dis 2005;41:45.
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12
Hepatitis C: Virological Aspects and Practical Implications
Antonio Alci Barone
Laboratory of Hepatitis, LIM 47 DMIP-HC-FMUSP; São Paulo, SP, Brazil
The hepatitis C virus (HCV) is a member of the Hepacivirus
genus of the family Flaviviridae.
Other important pathogens for humans, such as the
dengue virus and the yellow fever virus, belong to this family,
as do infectious agents in animals, such as the bovine viral
diarrhea virus and the classical swine flu virus.
The shape of HCV is spherical (50 nm in diameter), and it
has an enveloped nucleocapsid and a single-stranded RNA
genome of positive polarity. The HCV genome, with
approximately 10,000 nucleotides, consists of a single long
open reading frame (ORF) flanked by two noncoding regions
(NCRs) at the initial 5’ end, at which the internal ribosome
entry segment (IRES) is located, and at the 3’ end. Its
translation yields a large polyprotein (with nearly 3000 amino
acids) that is processed by viral and host cell proteases into
structural proteins, including the core, envelope 1 (E1), E2,
and p7 proteins, as well as the nonstructural proteins NS2,
NS3, NS4, and NS5. Recently, an alternate reading frame that
codifies an F protein with more than 160 amino acids has been
identified. However, its expression in natural HCV infection
has not been confirmed.
Structural proteins are cleaved by enzymes of the
parasitized cell. Envelope proteins are extensively glycosylated
and are involved in the binding with receptors as well as in
the entrance and fusion of the virus. The function of p7 protein
remains unknown. Nonstructural proteins, initially NS2 and
subsequently NS3, undergo self-cleavage and position
themselves in transmembrane domains across the host cell
membrane and into the cytosol or lumen (Figure 1).
Unlike that of the hepatitis B virus (HBV), the HCV genome
does not invade the infected cell nucleus. After the binding
through receptors (CD81, a tetraspanin, and the low density
lipoprotein receptor), the HCV genome acts directly as an
mRNA in the cytoplasm, where the translation is initiated
through the IRES in the 5’ NCR. The protein produced is
subsequently processed by the cell enzymes and by the
enzymes within the virus itself, yielding structural and
nonstructural proteins. After synthesis and maturation, these
nonstructural proteins and the RNA form replication complexes
that combine with the membrane and catalyze the translation
of intermediate negative strands of RNA, from which positivestrand progeny are generated. The genomic RNA and capsid
proteins unite, forming the nucleocapsid, which is transported
in cytoplasmic vesicles. While passing through the Golgi
complex, these vesicles assemble with the other particles and
undergo exocytosis and cell release (Figure 2).
The study of the HCV genome, even in samples obtained
from a single individual, reveals great heterogeneity among
the HCV genotypes. Genotypes in which multiple mutants
2007;11 (5) Suppl. 1:12-13.
coexist have been designated quasispecies. The multiple
mutations represent a rapid and very efficient mechanism for
the virus to evade the immune response and persist in the
host. The selection process and the process of adaptation
to the host have led to the evolution to different HCV
genotypes. The classification system most commonly used
is that proposed by Simmonds et al. and is based on the
similarity of the sequence of nucleotides using the following
criteria: similarity lower than 72% characterizes a new
genotype; similarity between 75 and 86% characterizes a
new subgenotype. There are 6 genotypes, which are
numbered from 1 to 6, with subgenotypes 1a, 1b, 1c, 2a, 2b,
2c, 3a, 3b, 4a, 5a, and 6a. Although the criterion is based on
molecular biology, this classification has practical,
pathogenetic, epidemiological, and treatment-related
implications. Therefore, subgenotype 1a is more prevalent
in the USA, 1b in Japan, 3a in Scotland, and 4a in Egypt/
Zaire. In Brazil, genotype 1 is found in approximately 60% of
the patients, followed by genotype 3, which is found in 20 to
30%, and genotype 2, which is found in a lower percentage.
Subgenotype 1b can cause severe forms of the infection
and, similar to genotype 4, does not respond as well to
treatment with IFN-α. Therefore, genotypes 1 and 4 should
be treated for 48 weeks.
The important advances in the knowledge of viral hepatitis
B and C are a consequence of some facts that will be discussed
herein.
The Use of Chimpanzees as a Model for the Study of Viral
Hepatitis
Although chimpanzees are not natural hosts for these
viruses, they reproduce the disease, thus allowing important
discoveries:
• Epidemiological studies: knowledge of these diseases
as communicable;
• Infectivity and titers of pools of HCV and HBV,
obtained from the infected animals;
• Infectivity of the molecular clones of HCV and
importance of genetic elements specific to HCV;
• Neutralization capacity of antibodies specific for HBV
and HCV;
• Protective immunity, tested through re-exposure to the
viruses;
• Mutants that escape to humoral and cellular immunity
could be recognized.
However, the use of nonhuman primates has advantages
and disadvantages.
Advantages
The only animal susceptible to the acute and chronic forms
of the disease; non-selected population; and sequential
biopsies.
www.bjid.com.br
Virological Aspects of Hepatitis C
Figure 1. Organization of the HCV genome, polyprotein
processing, and protein topology [1].
Figure 3. Systems used in the HCV in vitro study [1].
Disadvantages
Ethical considerations regarding the use of primates; cost
and availability; rarity of vertical transmission; more benign
disease presentation; and weaker, more limited immune
response
Use of Replicons
In HCV, subgenomic RNA replicons are those in which the
structural region has been replaced by the neomycin
phosphotransferase gene, and the translation of the
nonstructural proteins is regulated by the IRES of the
encephalomyocarditis virus. Through this experimental
approach, it became possible for the first time to effectively
and efficiently replicate HCV in vitro in cultured Huh-7 human
hepatoma cells. Interestingly, some simple amino acid
substitutions can increase the replication efficiency by up to
10,000 times in all of the nonstructural proteins. The replicon
system allowed the clarification of important aspects of the
virus life cycle, as well as simplifying the evaluation of new
antiviral strategies. However, some aspects of the virus life
cycle cannot be studied using this system.
13
Figure 2. HCV cycle in the host cell [1].
Efficient Cell Culture Systems for HCV
A 32-year-old male patient of Asian origin presented a
profile consistent with fulminant hepatitis. Using reverse
transcriptase polymerase chain reaction, HCV RNA was
detected in the serum during the acute phase and not during
the remission phase.
Using these samples, the complete HCV genome was
recovered and cloned. This strain, designated JFH1, has 9678
bp in the genome, with a single long ORF (nt 341-9439), and
encodes 3033 amino acids The transfection of this sample
into HUH-7.5.1 cultured cells allowed three independent
groups of researchers to obtain HCV infecting particles, for
tissue culture as well as for chimpanzee. These studies [2-4]
were published, between June and July of 2005, in the following
journals: Proceedings of the National Academy of Sciences,
Nature Medicine, and Science.
The infectious virions of HCV obtained in this way have
been used to infect laboratory animals and naïve cells. Such
infection can be monitored by the detection of the expression
of the NS5A, through analysis of reporter genes, or by direct
measurement of viral RNA (Figure 3).
In conclusion, more recent findings regarding the
virological aspects of HCV have greatly increased the
possibility of thoroughly studying this agent and especially
its relationship with the human host. Based on this approach,
it will be possible to better understand how to combat the
virus in chronically infected patients, thus preventing the
progression of the disease and its consequences.
References
1. Tellinghuissen T.L., et al. Studying Hepatitis C virus: making the
best of a bad virus. Journal of Virology 2007;81(17):8853-67.
2. Barthenschlager B., et al. Efficient hepatitis C virus cell culture:
what a difference the host cell makes. PNAS 2005;102(28):973940.
3. Wakita T., et al. Production of infectious hepatitis C virus in
tissue culture from a cloned viral genome. Nature medicine
2005;11(7):791-6.
4. Lindenbach B.D., et al. Complete replication of hepatitis C virus
in cell culture. Science 2005;309:623-6.
5. Kato T., et al. Sequence analysis of Hepatitis C virus isolated from
a fulminant hepatitis patient. J Med Virol 2001;64:334-9.
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14
Pathogenesis of Hepatitis C – HCV Consensus 2007
Ana Tereza R. Viso
The chronic hepatitis C virus (HCV) infects approximately
130 million people worldwide [1]. It is estimated that
approximately 15% of HCV-infected individuals eliminate the
virus spontaneously, that 25% develop a mild form of the
disease, and that 60% develop the chronic progressive form
[2]. The elimination or persistence of HCV infection depends
on the balance between the effectiveness, specificity and
rapidity of the innate and adaptive immune responses, as well
as on the HCV replication rate [3]. Persistence of HCV can
also be caused by infection at privileged (extrahepatic) sites,
viral inhibition of antigen presentation, selective immune
suppression, negative regulation of HCV gene expression,
viral mutations, immune exhaustion of T cells and the
incomplete differentiation of memory T cells [4,5].
Fibrosis is the principal complication of chronic hepatitis
C, and it is estimated that 20% of patients develop cirrhosis
over a period of 10, 20 or 30 years [2,6]. The progression of
fibrosis increases morbidity and mortality in chronic hepatitis
C [7], since it can lead to death due to complications caused
by cirrhosis or hepatocarcinoma [2].
Various studies have associated the progression of fibrosis
in hepatitis C with diverse factors such as: the kinetics and
pathogenicity of HCV; host-HCV interaction; intrinsic host
factors such as demographic profile, body mass index and
diabetes mellitus; host exposure to external factors; and the
form of HCV acquisition.
Life Cycle and Pathogenicity of HCV
Belonging to the Flaviviridae family, HCV is a small
enveloped virus [8]. Its genome consists of one RNA molecule
that is composed of two terminal regions, 5’- and 3’untranslated regions, and between these there is a single open
reading frame that encodes a polyprotein with approximately
3000 amino acids. This polyprotein cleaves at the N-terminal
side of three structural proteins, the nucleocapsid (core),
envelope 1 (E1) and envelope 2 (E2), all of which are involved
in the architectural organization of HCV. At the carboxylterminal side, the polyprotein cleaves to six nonstructural
proteins, NS2, NS3, NS4 (NS4A and NS4B), NS5 (NS5A and
NS5B) and NS6, which are responsible for the life cycle of the
virus [9].
After entering a susceptible host, HCV invades, infects
and replicates within the blood stream, repeating the process
in various tissues, as well as in peripheral B and T lymphocytes,
as it proceeds to the liver by tropism, passing through various
tissues such as those of the pancreas, thyroid, adrenal glands,
2007;11 (5) Suppl. 1:14-19.
spleen and bone marrow [10-12]. Since HCV can also directly
infect the lymphatic tissue, its stimulation can lead to the
development of B-cell lymphomas [13]. It is known that the
liver is the principal site of HCV replication, and various studies
have shown that this virus infects approximately 10% of
hepatic cells [5]. Infection with HCV at extrahepatic sites can
promote the appearance of HCV variants [14,15], thereby
decreasing the chance that the immune system will recognize
the virus.
To enter the host cell, HCV E2 and E1 proteins recognize
and bond with the CD81 receptors present on the surface of
hepatocytes and lymphocytes [16,17]. Circulating HCV
particles are accompanied by low-density and very lowdensity lipoproteins, which prompts discussion in the
literature regarding the possibility that low-density lipoprotein
is also a viral receptor [15]. After the interaction of the virus
envelope with the host cell membrane, HCV enters the cell
through endocytosis. In the cytoplasm, the messenger RNA
then undergoes translation, and polyproteins are processed;
the HCV RNA then replicates, after which the new viral ‘RNA’s
are packaged and transported to the surface of the host cell
so that they can disseminate and complete a new cycle [18].
The HCV replication rate is high, approximately 1 × 1012 virions
per day; this, together with its high mutation rate, estimated at
10-3 nucleotide substitutions per year, leads to great
heterogeneity in its presentations, which are known as
quasispecies [8]. The selection of and host adaptation to HCV
quasispecies have given rise to distinct genotypes [19] whose
classification is based on the similarity of the sequence of
nucleotides: similarity below 69% characterizes a new viral type;
and similarity between 75 and 80% characterizes a subtype [20].
The progression of fibrosis in chronic hepatitis C has been
associated with the diversity of HCV quasispecies [21]. The
production of new viruses is counterbalanced by the
destruction of infected cells through tissue apoptosis or
degradation in peripheral blood, since the half-life of the virus
in peripheral blood is approximately 2.7 hours [7]. Experimental
studies have shown that NS3 and NS5 proteins induce
apoptosis in infected hepatocytes [22].
In individuals infected with HCV, the persistence of the
virus can be attributed the large inoculum and the high rate of
viral replication, which allow the virus to evade the host
immune response [4,23]. There is controversy over whether
the sequence of nucleotides is directly associated with more
intense hepatic lesions [6,24,25].
There is some evidence of direct cytopathic lesion caused
by HCV, including HCV-induced histological lesions with scant
inflammatory infiltrate [26-28], fulminant hepatitis C after
chemotherapy in liver transplants [29] and HCV-related acute
cholestatic syndrome after renal transplantation [30].
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Pathogenesis of Hepatitis C
Studies suggest that specific genotypes, such as genotype
1, can be more cytopathic [31] or can induce more rapid
progression of the disease than do other genotypes [32].
Genotype 1 has been shown to be the genotype most strongly
associated with chronic HCV infection [33]. The risk of
cirrhosis and hepatocarcinoma has been shown to be greater
in individuals presenting genotype 1b than in those presenting
genotypes 2 and 3 [34]. However, other authors have stated
that HCV genotype and viral load do not influence the
progression of the disease [2,6].
It is known that steatosis is a cofactor that influences the
progression of fibrosis in chronic hepatitis C [35]. Various
studies have directly associated steatosis with HCV genotype
3 [36,37]. Therefore, genotype 3 is considered cytopathic
[38,39]. Kumar et al. identified the reduction of steatosis as
the only variable predictive of the virological response to the
treatment of chronic hepatitis C in individuals infected with
HCV genotype 3 [38].
Innate Immune Response to HCV
The innate immune response to HCV is responsible for
the activation of cytokines such as interferon (IFN) which
activate antiviral proteins that inhibit the replication of the
virus while the adaptive immune response to HCV neutralizes
viral particles and destroys infected cells [40]. Studies of HCVinfected chimpanzees that eliminate the virus without the
specific T-cell immune response suggest that, in some cases,
the innate immune response might be sufficient to destroy
the infection [41].
The RNA of HCV is recognized by the innate immune
response through the Toll-like receptor which responds with
the production of IFN-1α and IFN-1β [5,42]. IFN-1 stimulates
the nitric oxide synthase enzyme that is expressed in
hepatocytes and macrophages as the isoform inducible nitric
oxide synthase [43]. Patients with HCV who are treated with
IFN present higher levels of inducible nitric oxide synthase,
which have been correlated with lower serum levels of alanine
aminotransferase [44].
In addition, IFN-1 induces the production of various
proteins such as protein kinase (PKR), 2’,5’-oligoadenylate
synthetase (OAS) and the Mx protein [44]. These proteins are
responsible for the expression of the genes that inhibit the
replication of this virus within hepatocytes in an attempt to destroy
the infection [7,40]. When IFN bonds with the IFN receptor on
the surface of the infected cell, it activates the Janus kinase,
which induces phosphorylation of cytoplasmic proteins known
as signal transducers and activators of transcription (STATs),
specifically STAT 1 and STAT 2. The STATs form a dimer that
directs itself to the cell nucleus where it forms a complex with
the p48 protein, which is a stimulation factor for IFN-stimulated
gene factor 3. That complex bonds with the IFN-stimulated
response element ISRE, which is an RNA-polymerase promoter
complex, and there is a stimulus of the genes responsible for the
production of antiviral response proteins and of major
histocompatibility complex (MHC) proteins [44].
15
Some individuals present genetic alterations in the STATs
or in the Janus kinase that would impede the formation of
antiviral proteins [45].
Various viral proteins have shown a capacity to escape
the effect of IFN, as evidenced by the high rate of resistance
to treatment with IFN-α seen among individuals with hepatitis
C [44,45].
There are various characteristics of HCV that allow it to
evade the innate immune response:
• The viral replication complex appears to be composed
of a membrane that is highly resistant to in vitro
proteases and nucleases, which protects HCV from
detection by the innate immune response [40].
• The HCV core protein interacts with diverse cell factors,
including the tumor necrosis factor (TNF) receptor,
which decreases the cytolytic activity of T cells [46]
• Core proteins impede the antiviral activity of IFN, as
do NS3/4A and NS5A proteins [40].
• The NS3/4A proteins can impede the recognition of
the Toll-like receptor [5].
• The NS5A and E2 proteins can bind to PKR, thereby
blocking its activity [5].
• Multiple mutations in the IFN-sensitivity-determining
region (ISDR) modify the NS5A region, which inhibits
the phosphorylation of PKR, thereby impeding its
antiviral activity [45].
• The E2 region of HCV contains a sequence of eight
amino acids identical to those of PKR, and this
sequence is more common in genotype 1 than in
genotypes 2 and 3, which probably accounts for the
fact that individuals infected with genotype 1 present
greater resistance to treatment with IFN [44].
• Mutations in the ISDR sequence of NS5A suppress
the antiviral action of OAS.
• Levels of this protein are lower in nonresponders to
treatment with IFN [44].
The liver cell populations that participate in the innate
immune response are the natural killer (NK) cells, NK T cells,
Kupffer cells and dendritic cells [5]. The NK cells respond
minutes or hours after HCV infection by polarizing of the
granules in the direction of the infected cells as well as by
releasing perforins that fragment the nuclei of infected cells
and induce apoptosis [47]. They inhibit viral replication with
the production of IFN gamma (IFN-γ), which recruits intrahepatic
inflammatory cells and stimulates the T-helper 1 (Th1) response
[48], thereby inducing the necrosis or apoptosis of the HCVinfected cell [49]. Studies suggest that HCV inhibits receptor
genes in the activation of NK cells, decreasing the activity of
these cells by reducing their number and function in chronically
infected individuals [40]. The NK cells also have the capacity
to increase the functions of dendritic cells in the presence of
hepatic cells, although that capacity is impaired in NK cells
derived from patients with chronic hepatitis C, in which the
production of interleukin (IL)-10 and transforming growth factor
beta (TGF-β) can inhibit the activity of dendritic cells [50].
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16
After HCV enters the host cell, the binding of the E2
glycoprotein with the CD81 receptor of NK cells inhibits the
function of the NK cells [7,51], which alters the immune
response to HCV infection. The E2 glycoprotein also inhibits
cytotoxicity and the production of IFNγ by NK cells [40].
Various studies have suggested that the failure of dendritic
cells to recognize HCV contributes to the persistence of
hepatitis C [50,52-54].
Humoral Immune Response to HCV
After HCV infection, there is expression of the
hypervariable NS1/E2 region on the surface of the virus, which
stimulates B cells to produce high antibody titers of antibodies
with the objective of destroying the permanence of the virus
[44]. The appearance of anti-HCV antibodies is significantly
delayed, and these antibodies can first be detected from 7 to
31 weeks after infection [7]. The host applies selective pressure
on HCV, and this stimulates high nucleotide variation, as well
as the appearance of mutations in the envelope proteins, from
which the virus selects genomic variants in an attempt to
eliminate the site of immune response recognition [55]. The
great quantity of HCV quasispecies formed allows the virus
to evade the humoral immune response, and the effect of HCVneutralizing antibodies appears to be insufficient to control
the infection [7], which therefore persists [15].
Similar to what occurs in auto-immune type 2 hepatitis,
HCV can mimic the immune system, leading to viral escape or
postinfection immunity [4,56]. Anti-HCV antibodies have been
implicated in tissue damage due to the formation of
immunocomplexes such as antinuclear antibodies [57], autoantibodies that act against cytochrome P450 and antibodies
that act against the liver and kidney [4]. The deposition of
immunocomplexes has been related to the appearance of
extrahepatic manifestations, such as arthritis,
cryoglobulinemia [58], vasculitis, glomerulonephritis, Sicca
syndrome and itchiness, all of which cause considerable
morbidity [57].
There is evidence that HCV infection can be resolved by
the cell response with specific CD4+ and CD8+ T cells when
there is no formation of antibodies against this virus [59,60],
showing that the humoral immune response is not always
involved in the response to HCV infection.
Cell Response to HCV
Since there is a weak humoral immune response to HCV, it
is believed that the reactivity of cytotoxic T-lymphocytes
(CTLs) or CD8+ T cells is fundamental to viral elimination
[61,62], and that impairment of this reactivity is one of the
factors responsible for the chronicity of the infection
[7,63,64]. The CD8+ T cells can eliminate HCV from the liver
through two mechanisms: inducement of apoptosis in
infected hepatocytes; and suppression of replication by the
production of IFN-γ [22,65]. The CTL response is less
vigorous in chronically infected patients than in those
presenting acute infection [4]. This can be the result of
immunologic tolerance or exhaustion of the CD8+ T cell
response to the high viral load that persists in individuals
chronically infected with HCV [4].
In addition to CD8+ T cells, CD4+ T cells seem to be
involved in the viral damage mediated by the increased
expression of MHC class II molecules. Some studies have
attributed the vigorous and long-lasting response of CD4+ T
cells to the elimination of HCV in the acute form the infection
[4,66]. However, the loss of the specific CD4+ T cell reactivity
to HCV has been associated with the persistence of the virus
and the progression of liver damage [67,68].
In acute HCV infection, the peak in serum levels of
transaminases corresponds with the cell response, which
suggests that the hepatic lesion is immune-mediated [5,64]. It
is known that, after activation, T cells initiate clonal
proliferation by secreting cytokines and other substances that
can affect hepatic function in a variety of ways [69].
Various cytokines act as mediators in the inflammation
caused by chronic hepatitis C and have been related to
hepatocyte death, i.e. cholestasis and fibrosis, and
paradoxically play a role in regeneration following hepatic
injury [69,70]. It is argued that the imbalance between the
production of Th1 and Th2 cytokines is related to the
progression of chronic hepatitis C. The expression of Th1
cytokines such as IL-2 and TNF-α has been shown to be
related to the more aggressive presentation of hepatic disease,
whereas the expression of Th2 cytokines such as IL-10 has
been shown to be related to the milder presentation [71].
The production of TNF-α is one of the earliest events in
hepatic injury and is the ‘trigger’ for the production of other
cytokines [72], as well as being implicated in the inducement
of hepatocyte apoptosis in viral hepatitis [73].
The levels of cytokines such as IFN-γ, TNF-α, IL-6 and
IL-8 are elevated in individuals with chronic hepatitis C [7482], and some authors have shown that this increase is
proportional to the extent of the damage, histologically
[26,76,83,84].
There is evidence that IL-4 can modulate the immune
response in HCV-infected individuals [75], principally through
the activity of Th2 cells.
It has been shown that IL-10 can suppress proliferation in
the Th1 and Th2 responses, as well as inducing anergy [85].
There is evidence that IL-10 levels increase in chronic hepatitis
C [75]. Some studies report reduced inflammatory activity [86],
and others report that administration of IL-10 to such patients
causes fibrosis [87].
Various studies have shown that TGF-β is increased in
chronic hepatitis C and is involved in the progression of
fibrosis, which has been challenged by other authors [88,89].
It has been suggested that TGF-β and IL-10 act as
immunosuppressive agents in the liver [90]. In addition, both
have been shown to inhibit the immune response and regulate
the activity of dendritic cells [91], which can establish a
balance between the Th1 and Th2 responses in chronic
diseases [92].
www.bjid.com.br
Host Factors Associated with the Persistence and
Progression of Hepatitis C
In HCV infection, the genetic constitution and immune
‘status’ of the host are important factors in the persistence
and progression of the virus [23,93], since they influence
antigen recognition and presentation, as well as the type of
Th response [94].
Some MHC class II alleles, such as DR5, have been
associated with a lower incidence of cirrhosis in individuals
chronically infected with HCV [94]. Rehermann et al. [95]
identified CTLs restricted by histocompatibility leukocyte
antigen A2 in 97% of chronic hepatitis C patients, compared
with 2% of anti-HCV-negative controls. It is speculated that
the MHC class II molecule presentation of antigens is deficient
in HCV-infected cells, since some viral proteins inhibit the
presentation of the antigen through IFN-induced negative
immunoregulation [44].
Some pro-inflammatory cytokines appear to be associated
with the viral infection response as well as with the expression
of specific haplotypes [94], such as IL-10 haplotypes, which
can be predictors of spontaneous elimination of HCV [96].
However, there is disagreement in the literature, since other
authors did not find evidence for polymorphism in the studied
genes being considered as a relevant factor in the elimination
of HCV or in the response to treatment [97-99].
The influence of demographic data such as age, gender
[6,34] and ‘race’ [100,101] in the progression of hepatitis C
can be due to genetic variations existent among those. Some
studies report that HCV positivity increases with age [34,101],
thereby leading to a greater chance of progression of the
disease [6,34]. The male gender is more prevalent in most
studies on hepatitis C [6,101] and, in addition, it was associated
with the progression of the disease to cirrhosis [6,34]. Some
studies suggest that Afro-Americans, due to a greater
propensity to chronicity, resistance to treatment (higher
percentage of genotype 1) and development of
hepatocarcinoma, present a worse evolution of hepatitis C
than do Caucasian-Americans [100,102]. Analyzing 99 chronic
HCV-infected individuals and 31 individuals who had
spontaneously eliminated HCV, Sugimoto et al. [103] found
evidence that the CD4+ T-cell response was less vigorous in
Afro-Americans than in Caucasian-Americans, with a
predominance of the Th2 response and maintenance of the
infection. The evolution of hepatitis C in different ethnicities
could be due to genetic factors, such as the presence of HLA
class II alleles, which could define the spontaneous elimination
of HCV [104].
There are various extrinsic host factors that are related to
the progression of chronic hepatitis C: alcohol abuse; smoking
[6,34,105-109]; the endovenous acquisition of HCV; and
coinfection with other viruses such as HIV, HBV and human
T-cell lymphotropic virus [6,110,111].
The prevalence of HCV infection is higher among
individuals who consume alcohol [112,113]. Studies suggest
that alcohol increases the ability of HCV to enter and persist
17
within the organism [112]. Other studies argue that alcohol
intake affects some components of the immune response
[112] and can alter the inflammatory response of cytokines,
thereby increasing viremia, which can be an important
cofactor in the development of hepatocarcinoma [114]. In
addition, alcohol intake in HCV-infected individuals
increases hepatic steatosis and induces apoptosis
[107,112,115].
Smoking, in addition to increasing inflammatory activity
and hepatic fibroses [109], can induce direct injury to the
liver, as well as causing indirect damage (toxic effect), and
can have immunological effects (production of IL-1, IL-6 and
TNF-a, which cause liver damage).
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19
20
Antifibrotic Therapy in Chronic Hepatitis C
Rinaldo Focaccia Siciliano and Antonio Alci Barone
The most pronounced histological characteristics of
chronic hepatitis C are hepatocellular necrosis, inflammatory
infiltrate and fibrosis. Unlike the first two, which present
fluctuations, the evolution of liver fibrosis progressively
evolves toward pathological sequelae due to the chronic liver
injury induced by hepatitis C virus (HCV). Fibrosis results
from the accumulation of extracellular matrix components,
which leads to distortion of the liver architecture, alterations
in hepatic microcirculation, and cellular dysfunction. This
pathological hepatic process develops slowly and
progressively, producing clinical repercussions only in its final
stage, liver cirrhosis, which can take decades. Therefore,
determination of the degree of fibrosis through liver biopsy
or progression rate is crucial to the understanding of the natural
history of chronic hepatitis C.
Some authors have identified host factors that are
associated with the accelerated progression of liver fibrosis
in HCV infection. The most important are being over 40 years
of age at the time of HCV infection, being male, consuming
excessive quantities of alcohol, and presenting conditions
that lead to immunodeficiency, such as HIV infection or
transplant. Other factors also seem to contribute to a more
rapid progression toward fibrosis, such as liver steatosis, coinfection with hepatitis B virus (HBV), obesity, and diabetes
mellitus.
Recent advances in clinical studies and basic science have
brought new perspectives to the development of therapies
that can curb the progression of the fibrogenic process or
even promote reversion of liver fibrosis. These advances
occurred after the recognition of fibrogenic cell types in the
liver, as well as of the principal sites of fibrosis formation and
its reversibility potential.
Fibrosis seems to result from an imbalance between the
synthesis and degradation of the extracellular matrix, resulting
in the accumulation of conjunctive tissue in the liver. This
process is triggered and sustained by the chronic liver damage
caused by HCV and leads to the disruption of the normal liver
architecture, culminating in the development of cirrhosis/
hepatic insufficiency. Stellate cells are the principal source of
extracellular matrix in the damaged liver tissue. In a normal
liver, they are present in the Disse space and are the principal
reservoirs of vitamin A. Chronic hepatic injury secondary to
HCV, through the increase of free radicals and fibrogenic
mediators, leads to the activation of stellate cells, as well as to
their proliferation and differentiation into myofibroblasts, when
they acquire contractile, pro-inflammatory and fibrogenic
properties. Once activated, they migrate to the sites of injury
2007;11 (5) Suppl. 1:20-21.
with the objective of effecting repair by secreting a large
quantity of extracellular matrix. Activated stellate cells also
produce cytokines that perpetuate their activation status, as
well as being stimulated by the apoptosis of damaged
hepatocytes. The leukocytes attracted into the hepatic
inflammatory process secondary to HCV infection also induce
the production of collagen by stellate cells. These activated
cells, in turn, have a pro-inflammatory effect that feeds a
vicious cycle.
Under physiological conditions, the excess extracellular
matrix is degraded through regulation by metalloproteinases.
Activated hepatic myofibroblasts, in addition to producing
large quantities of Type I and Type III collagen, secrete tissue
inhibitors of metalloproteinases, which block the
collagenolytic activity. We have begun to comprehend various
parts of this process of accumulation of extracellular matrix in
the liver tissue, although there are still many gaps in our
understanding. In addition to the efficient collagenolytic
activity of the metalloproteinases, the degradation of the
extracellular matrix in the liver can also occur through the
activities of neutrophils, macrophages and stellate cells
themselves; however, the importance and modulation of each
have yet to be clarified. The activation of stellate cells, as well
as their proliferation and fibrogenic activity, is regulated by
various soluble growth factors, such as platelet-derived
growth factor, transformation growth factor beta, and
endothelin-1.
Determining the mechanisms involved in the process of
liver fibrosis has led to a new perspective on the development
of antifibrotic drugs in animal models. However, there is still
an obstacle to be transposed before positive laboratory results
can be reproduced in humans. New lines of research attempt
to attenuate the activation of stellate cells, inhibit some of
their properties when activated, promote their apoptosis, or
stimulate the degradation of the extracellular matrix. No specific
antifibrotic therapies have been approved for use in humans;
however, considerable effort has been made in laboratory
studies in this promising area.
In clinical practice, the treatment of hepatitis C with the
combination of pegylated interferon alpha and ribavirin is the
only pharmacological regimen currently available that can
modify the natural evolution of liver fibrosis. This does not
simply occur by suppressing the viremia (achieving a
sustained virological response) but possibly by the antifibrotic
role of pegylated interferon alpha. Recent clinical studies show
remission of liver fibrosis or reduction of its progression rate
in patients under treatment with therapeutic regimens
containing interferon alpha or pegylated interferon. This effect
is even more apparent among hepatitis C patients presenting
a biochemical or virological response after therapy. Notably,
www.bjid.com.br
Antifibrotics and Chronic Hepatitis C
most of these studies are retrospective, and their principal
objective was the evaluation of the virologic response to the
treatment with interferon alpha.
Although liver fibrosis is a dynamic process, its remission
is slow and demands prolonged therapy and follow-up
evaluation. Multicentric prospective comparative studies that
evaluate the histological impact of maintenance treatment with
pegylated interferon in low doses versus placebo in patients
with hepatitis C who are nonresponsive to treatment are
currently being carried out. Preliminary results of these
studies are quite promising; reduction of fibrosis or clinical
complications resulting from liver cirrhosis can be seen after
approximately two years of therapy with pegylated
interferon. The final results, with histological analysis, after
long-term follow-up treatment and inclusion of a large
number of patients, will consolidate an evaluation of safety,
clinical and histological benefits, and cost-effectiveness ratio
of the prolonged use of pegylated interferon in low doses in
hepatitis C.
The role of the physician in the positive modification of
the natural history of hepatitis C-related liver fibrosis should
not be restricted to the removal of HCV as a hepatic aggressive/
pro-inflammatory factor (achieving a sustained virologic
response) or to the use of interferon as a potential antifibrotic
agent. In daily practice, other strategies aimed at attenuating
the progression of liver fibrosis are based on the effects on
modifiable factors that can alter the natural history of HCV:
21
drinking cessation; prevention and control of obesity; and
prevention of HIV or HBV co-infection.
Much progress has been achieved in basic science
regarding the understanding of the biological mechanisms
that lead to the development of liver fibrosis. However, clinical
trials to validate new drugs or antifibrotic strategies are
warranted.
References
1. Albanis E, Friedman S,L. Antifibrotic agents for liver disease. Am
J Transplant 2006;6(1):12-9.
2. Friedman S.L., Rockey D.C., Bissell D.M. Hepatic fibrosis 2006:
report of the Third AASLD Single Topic Conference.
Hepatology 2007;45(1):242-9.
3. Bataller R., Brenner D.A. Liver fibrosis. J Clin Invest
2005;115(2):209-18.
4. Marcellin P., Asselah T., Boyer N. Fibrosis and disease progression
in hepatitis C. Hepatology 2002;36(5 Suppl 1):S47-56.
5. Friedman S.L., Bansal M.B. Reversal of hepatic fibrosis - fact or
fantasy? Hepatology 2006;43(2 Suppl 1):S82-8.
6. Everson G.T., Hoefs J.C., Seeff L.B., et al. Impact of disease severity
on outcome of antiviral therapy for chronic hepatitis C: Lessons
from the HALT-C trial. Hepatology 2006;44(6):1675-84.
7. Kaiser P., Hass H., Lutze B., et al. Long-term low dose treatment
with pegylated interferon alpha 2b leads to a significant reduction
in fibrosis and inflammatory score in chronic hepatitis C
nonresponder patients with fibrosis or cirrhosis. 57th Annual
Meeting of the American Association for the Study of Liver
Diseases. Massachusetts – USA 2006.
8. Afdhal N., Freilich B., Levine R., et al. Colchicine versus peginterferon long-term (COPILOT) trial: interim analysis of clinical
outcomes at year 2. Hepatology 2004;40:238A.
www.bjid.com.br
22
Laboratory Testing for Hepatitis C
Neiva Sellan Lopes Gonçales and Fernando Lopes Gonçales Junior
Hepatitis Study Group – MI/FCM/UNICAMP; Campinas, SP, Brazil
Serological Detection of Hepatitis C Virus
Serological diagnosis of patients infected with the hepatitis
C virus (HCV) can be performed using two categories of tests:
indirect tests, which detect antibodies against HCV; and direct
tests, which detect, quantify, or characterize components of
the viral particle, such as HCV RNA testing and testing for
detection of the HCV core antigen.
Anti-HCV antibodies are usually detected using third- and
fourth-generation immunoenzymatic assays – enzyme
immunoassay (EIA)/enzyme-linked immunosorbent assay
(ELISA) 3 and EIA/ELISA 4, respectively – which contain
HCV core antigens and HCV nonstructural genes. The
specificity of the EIA tests available on the market that detect
anti-HCV was determined to be higher than 99%, whereas
their sensitivity, which was more difficult to determine due to
the lack of gold standard tests with high sensitivity, was 9599% [1]. However, false-positive results for anti-HCV can
occasionally occur, especially in populations with prevalence
rates below 10% [2-4].
There are many reasons why laboratories do not routinely
use a supplementary test based on immunoblot analysis, such
as the recombinant immunoblot assay, to complement the
diagnosis of HCV infection. In addition to the high cost of
such a test, the lack of laboratory standards that can evaluate
its performance and interpretation, in conjunction with its
actual accuracy, is among the principal reasons. Furthermore,
this type of test does not distinguish past from present
infection, and its use is only indicated for confirmation of EIA
results.
In contrast, the use of nucleic acid testing (NAT) makes it
possible to differentiate between viremic and nonviremic
individuals by detection of HCV RNA, allowing the clinician a
differentiated approach to anti-HCV-positive individuals.
However, there can be situations in which HCV RNA is not
detected (negative HCV RNA) and the individual has active
infection with HCV. This can occur in individuals in whom
anti-HCV antibody titers are high and RNA titers are low [5].
Therefore, HCV RNA might not be detectable in certain
individuals in the acute phase of the disease. However, these
findings are transient, and chronic infection can develop [6].
In addition, HCV RNA intermittent positivity has been
observed in individuals chronically infected with HCV [6-8].
Negativity of HCV RNA results can indicate resolved infection.
In 15 to 25% of those anti-HCV positive individuals who
acquired the infection after 45 years of age, the infection
resolves spontaneously. This percentage increases to 40-45%
in those who acquired the HCV infection in childhood or
young adulthood [9].
Different tests based on polymerase chain reaction (PCR)
have been developed to directly detect the viral particle. One
characteristic of real-time PCR is amplification coupled with
2007;11 (5) Suppl. 1:22-24.
detection, which allows the evaluation of the number of viral
genomes at the onset of and throughout the reaction.
Qualitative detection of HCV RNA by reverse transcriptase
(RT)-PCR is generally accepted as the most sensitive and
standardized test to date [10,11]. Nevertheless, there is variability
among the results from different laboratories, as evidenced by
the use of international panels of proficiency. The accuracy
and reliability of the results are directly related to the laboratory
procedures adopted in the performance of the tests [12]. The
lack of preliminary care in sample collection, in conjunction
with the time involved in preparing and separating the samples,
can result in incorrect results. It is extremely important that all
laboratory procedures comply with Good Laboratory Practice
and strictly follow the protocols standardized by the
manufacturers of the diagnostic kits and reagents.
The gold standard consists of the careful use of NAT,
standardized for detection of HCV RNA, together with EIAs
(specificity in conjunction with sensitivity).
An alternative to aid diagnosis is the use of the ratio
between optical density and cut-off value (OD/COV) or the
sample/cut-off ratio as an indicator of the true positivity of
the test. Studies carried out in Brazil show that, in EIAs,
reagents with OD/COV greater than 3 are repeatedly associated
with 100% true-positive results (positive predictive value) and
present approximately 92% positivity for HCV RNA by RTPCR [13]. In terms of the population studied, the positive
predictive value is increased when accompanied by risk
factors, high levels of alanine aminotransferase (ALT), or liver
disease.
In immunocompetent patients, EIAs present excellent
reproducibility; however, in hemodialyzed or
immunocompromised patients, EIA sensitivity is significantly
reduced [14].
In low-risk populations, such as blood donors, or in
random population screening, i.e., in populations that do not
present risk factors for the acquisition of HCV infection,
negative EIA results are sufficient to rule out the presence of
HCV. However, false-positive results can occur in these
populations. In such cases, a qualitative study of HCV RNA
should be performed to confirm the diagnosis.
In high-risk populations, when there is clinical suspicion of
HCV infection, positive EIA results confirm the exposure to
HCV. A qualitative study of HCV RNA should be performed to
distinguish individuals with chronic infection from those who
have eliminated the HCV spontaneously.
In patients with chronic hepatitis of unknown cause and
negative anti-HCV EIA results, especially in
immunocompromised patients [14], a qualitative study of HCV
RNA should be performed. The presence of HCV RNA confirms
the diagnosis, although a negative result does not rule out
HCV infection. In such cases, it is recommended that a new
HCV RNA study be performed six months after the first study.
Detection of the HCV core antigen by EIA can be an alternative
for early diagnosis of HCV infection.
www.bjid.com.br
The HCV core antigen ELISA was developed to be used
as a serological screening test to detect the HCV core antigen,
especially during the immunological window period, when
antibodies are not detected. This assay was found to have
sensitivity close to that of NAT, with a mean difference in
detection of one to two days [15].
Based on this assay, a new assay was developed to detect
and quantify HCV core antigen. The modifications made to
this new assay, such as the dissociation of immune complexes,
which allows the detection of free antigens and core antigen
antibodies, and the change in the signal amplification, through
the modification of the conjugate, have increased the
sensitivity of the test. Studies have demonstrated that this
test can reduce the immunological window by 3.3 days in
comparison with the previous test (i.e., the HCV core antigen
ELISA). This increase in sensitivity has led to a significant
(58-day) decrease in the size of the immunological window.
The difference between this EIA and PCR was only 0.24
days [16].
This test can be considered a viable alternative to
detecting viremia directly when NAT cannot be used for
reasons of cost, organization, emergency, or logistic difficulties.
Tests that allow simultaneous or combined detection of HCV
core antigen and antibody in a single assay are currently
available on the market. These tests, known as HCV Ag/Ab
combo assays, have high sensitivity and specificity, reducing
the duration of the immunological window (during which
antibodies are not detected) by up to 12 days [17]. Studies
carried out using this assay showed sensitivity close to that
of NAT, with a mean difference in detection of 1 to 2 days [18].
The use of NAT in the diagnosis of HCV infection makes it
possible to distinguish viremic from nonviremic individuals
through the detection of HCV RNA.
Therefore, these tests can be considered a plausible future
solution in the screening of blood donors, organ
transplantation programs, and cases of occupational exposure,
in which a rapid and low-cost diagnosis is necessary.
In order to standardize the tests, the World Health
Organization and the United States National Institute for
Biological Standards and Controls have established a standard
measure known as the international unit (IU). Assays for
qualitative detection of HCV RNA are important tools because
they are significantly more sensitive than are most quantitative
tests. Qualitative assays are based on the principle of target
amplification using either PCR or transcription-mediated
amplification. The cut-off value of the lower limit of detection
of HCV RNA of these commercial assays is 50 IU/mL and 6 IU/
mL, respectively [19]. The specificity of these essays exceeds
99%. A positive test for HCV RNA confirms active replication
of HCV. Clinical and laboratory follow-up with study of HCV
RNS should be performed to confirm the absence of active
replication of HCV. Once HCV infection is confirmed,
performing further qualitative tests for HCV RNA in patients
submitted to clinical follow-up evaluation but not receiving
treatment has no diagnostic utility.
The quantification of HCV RNA can be performed by
target amplification using PCR or by signal amplification using
branched DNA (bDNA). In these commercial assays, the cutoff value of the lower limit of quantification of HCV RNA
ranges from 600 to 615 IU/mL, and the upper linear limit ranges
from 850,000 to 7,700,000 IU/mL [20]. The standardization in
IU does not represent the actual number of viral particles in
the preparation. There are significant variations among
commercial assays. The dynamics of each assay should be
observed, and appropriate dilutions of the material being
analyzed should be performed to ensure the accuracy of the
quantification.
The ideal assay for HCV RNA should have a lower
detection limit of 5 to 50 IU/mL and a linearity curve of 6 to 7
log10. Traditional assays for detection of viral load, such as
bDNA and Roche Monitor, present detection limits of 615 IU/
mL and 600 IU/mL respectively [21,22], which are inadequate
to define end-of-treatment response or sustained virological
response. Real-time PCR assays are a promising tool due to
their sensitivity and broad range of linearity. Cobas Taqman
48 HCV assay is a quantitative assay that has a detection limit
of 10 to 100 IU/mL, which makes it well suited for use in followup treatment (at the initiation and at week 12) [23].
23
Acute Infection and Cutting/Piercing Accidents
After exposure to HCV, anti-HCV antibodies can be
detected by EIA in 50 to 70% of the patients at the onset of
symptoms, this percentage increasing to approximately 90%
after 3 months. Routinely, HCV RNA can be detected between
post-exposure weeks 1 and 3, remaining at detectable levels
when symptom onset occurs. From post-infection week 2 to
post-infection week 8, levels of ALT rise, and this increase is
accompanied by the appearance of hepatocytic lesions.
Vertical Transmission
An important question is that of exactly how mother-tochild transmission of the HCV infection is defined. In many
children born to mothers with chronic hepatitis C, anti-HCV
(IgG) is detectable in the blood. These antibodies are acquired
through passive transplacental transfer. These passively
acquired antibodies will remain detectable for the first 12 to 15
months of life. Therefore, the criterion to identify mother-tochild transmission of HCV infection is the detection of antiHCV and HCV RNA in the blood of the child after the age of 18
months.
Chronic Infection
In patients with chronic hepatitis C, the diagnosis of
chronicity is based on the detection of anti-HCV and HCV
RNA in the blood, using techniques of high sensitivity, and is
confirmed through liver biopsy.
Loss of anti-HCV and isolated presence of HCV RNA are
uncommon in immunocompetent patients with chronic
hepatitis C. However, these findings can occur in
hemodialyzed patients and in severely immunocompromised
patients
Follow-Up Treatment
Some patients with detectable HCV RNA should be
considered for treatment. Genotyping should be performed
at the initiation of treatment in order to define treatment
duration, since, according to treatment protocols, patients
infected with genotype 2 or 3 should be treated for 24
www.bjid.com.br
24
weeks, whereas those infected with genotype 1 should be
treated for 48 weeks [24].
A considerable limitation in the evaluation of patients
with chronic infection with HCV has been the lack of
standardization of the tests for detection of HCV RNA. A
significant difference has been observed in the assays used,
both in terms of sensitivity (upper limit of detection) and in
terms of dynamics. These differences are observed not only
among the different assays but also among different
laboratories performing a given assay. Therefore, it is
important that, throughout the clinical follow-up of a patient
receiving specific treatment, the same assays and, if
possible, the same laboratory always be used [25-28].
Quantification of HCV RNA should be performed in
the pretreatment sample and in the week-12 sample in
order to evaluate the predictive value of the treatment
response.
Since the qualitative study of HCV RNA presents a
lower limit of detection of 50 IU/mL, it should be used at
week 4 of treatment, as a predictor of sustained virologic
response (SVR), then again, to detect the SVR, at the
end of treatment and at 6 months after the end of
treatment. Therefore, presenting negative PCR results
by week 4 of treatment has a high predictive value for
achieving an SVR.
13. Gonçales N.S.L., Costa F.F., Vassalo J., Gonçales Jr., F.L. Diagnosis
of hepatitis C in Brazilian blood donors using a reverse
transcriptase nested polymerase chain reaction: comparison
with enzyme immunoassay and recombinant protein immunoblot
assay. Rev Inst Med trop S Paulo 2000;42(5):263-7.
14. Lakshmi V., Reddy A.K., Dakshinamurty K.V. Evaluation of
commercially available third-generation anti-hepatitis C virus
enzyme-linked immunosorbent assay in patients on
haemodialysis. Indian J Med Microbiol 2007;25:140-2.
15. Tanaka E.C., Ohue K., Aoyagi K., et al. Evaluation of a new
enzyme immunoassay for hepatitis C virus (HCV) core antigen
with clinical sensitivity approximating that of genomic
amplification of HCV RNA. Hepatology 2000;32:388-93.
16. Laperche S., Le Marrec N., Simon N., et al. A new HCV core
antigen assay based on disassociation of immune complexes: an
alternative to molecular biology in the diagnosis of early HCV
infection. Transfusion 2003;43:958-62.
17. Laperche S., Elghouzzi M.-H., More P., et al. Is an assay for
simultaneous detection of hepatitis C virus core antigen and
antibody a valuable alternative to nucleic acid testing?
Transfusion 2005;45:1965-72.
18. Laperche S., Le Marrec N., Girault A., et al.. Simultaneous detection
of hepatitis C virus (HCV) core antigen and anti-HCV antibodies
improves the early detection of HCV infection. J Clin Microbiol
2005;43:3877-83.
19. Desombere I., Van Vlierberghe H., Couvert S., et al. Comparison
of qualitative (COBAS AMPLICOR HCV 2.0 versus VERSANT
HCV RNA) and quantitative (COBAS AMPLICOR HCV monitor
2.0 versus VERSANT HCV RNA 3.0) assays for hepatitis C
virus (HCV) RNA detection and quantification: impact on
diagnosis and treatment of HCV infections. J Clin Microbiol
2005;43(6):2590-2.
20. Elbeik T, Surtihadi J, Destree M et al. Multicenter evaluation of
performance characteristics of the Bayer VERSANT HCV RNA
3.0 assay (bDNA). J Clin Microbiol 2004;42:563-9.
21. Ve i l l o n P. , P a y a n C . , P i c c h i o G. , e t a l . C o m p a r a t i v e
evaluation of the total hepatitis C virus core antigen,
branched-DNA, and Amplicor Monitor assays in
determining viremia for patients with chronic hepatitis C
during interferon plus ribarin combination therapy. J Clin
Microbiol 2003;41:3212-20.
22. Nolte F.S., Fried M.W., Shiffman M.L., et al. Prospective
multicenter clinical evaluation of AMPLICOR and COBAS
AMPLICOR hepatitis C virus tests. J Clin Microbiol
2001;39:4005-12.
23. Konnick E.Q., Willians S., Ashwood E.R., et al. Evaluation of
Cobas hepatitis C virus (HCV) Taqman anlyte –specific reagent
assay nd comparison to the Cobas Amplicor HCV monitor v2.0
and Versant HCV bDNA 3.0 assays. J Clin Microbiol
2005;43:2133-40.
24. Fried M.W., Shiffman M.L., Reddy C., et al. Peginterferon alfa-2a
plus ribavirin for chronic hepatitis C virus infection. N Engl J
Med 2002;347:975-92.
25. Shiffman M.L., Ferreira-Gonzalez A., Reddy K.R., et al.
Comparison of three commercially available assays for HCV
RNA using the International Units standard: Implications for
management of patients with chronic hepatitis C virus infection
in clinical practice. Am J Gastroenterol 2003;98:1159-66.
26. Podzorski R.P. Molecular testing in the diagnosis and management
of hepatitis C virus infection. Arch Pathol Lab Med
2002;126:285-90.
27. Schirm J., van Loon A.M., Valentine-Thon E., et al. External
quality assessment program for qualitative and quantitative
detection of hepatitis C virus in diagnostic virology. J Clin
Microbiol 2002;40:2973-80.
28. Pawlotsky J.M. Diagnostic testing in hepatitis C virus infection:
viral kinetics and genomics. Semin Liver Dis 2003;23:3-11.
References
1. Alter M.J., Kuhnert W.L., Finelli L. Guidelines for Laboratory
Testing and Result Reporting of antibody to hepatitis C Virus.
MMWR 2003;52:1-15.
2. Kleinman S., Alter H., Bush M., et al. Increased detection of hepatitis
C virus (HCV) – infected blood donors by a multiple-antigen HCV
enzyme immunoassay. Transfusion 1992;32:805-613.
3. Conry-Cantilena C., VanRaden M., Gibble J., et al. Route of infection,
viremia, and liver disease in blood donors found to have hepatitis
C virus infection. N Engl J Med 1996;334:1691-6.
4. Hyams K.C., Riddle J., Rubertone M., et al. Prevalence and
incidence of hepatitis C virus infection in the US military: a
seroepidemiologic survey of 21000 troops. Am J Epidemiol
2001;153:764-70.
5. Busch M.P., Kleinmam S.H., Jackson B., et al. Nucleic acid testing
of blood donors for transfusion-transmitted infectious diseases:
report of Interorganization Task Force on Nucleic Acid
Amplification of Blood Donors. Transfusion 2000;40:143-59.
6. Thomas D.L., Astemborski J., Rai R.M., et al. Natural history of
hepatitis C virus infection: host, viral, and environmental
factors. JAMA 2000;284:450-6.
7. Alter M.J., Margolis H.S., Krawczynski K., et al. Natural history
of community-acquired hepatitis C in the United States. N Engl
J Med 1992;327:1899-905.
8. Larghi A., Zuin M., Crosignani A., et al. Outcome of an out break
of acute hepatitis C among healthy volunteers participating in
pharmacokinetics studies. Hepatology 2002;36:993-1000.
9. Alter H.J., Seef L.B. Recovery, persistence, and sequelae in hepatitis
C virus infection: a perspective on long term outcome. Semin
Liver Dis 2000;20:17-35.
10. Pawlotsky J.M. Molecular diagnosis of viral hepatitis.
11. Pawlotsky J.M. Use and interpretation of virological tests for
hepatitis C. Hepatology 2002;36:S65-S73.
12. Callendo A.M., Valsamakis A., Zhou Y., et al. Multilaboratory
comparison of hepatitis C virus viral load assays. J Clin Microbiol
2006;44:1726-32.
www.bjid.com.br
25
Hepatitis C: Genotyping
Norma de Paula Cavalheiro
The heterogeneity of the hepatitis C virus (HCV) genome
was described at the beginning of the 1990s. Subsequently,
genotyping tests were developed in order to delineate and
differentiate these variants, leading to various classifications
in different parts of the world. However, in 1994, in a consensus
publication, criteria were established and a universal
classification was consolidated, comprising the identification
of 6 large groups or viral genotypes, in addition to over 70
different subtypes distributed worldwide. It was established
that genotypes differ from one another in 31% to 33%, as do
the subtypes in 20% to 25%.
The standardization in HCV classification was in
accordance with the uniformity of data published in scientific
and epidemiological studies on evolution and pathogenesis.
Due to genomic peculiarities of different populations in the
world, databases for centralization and collection of
information on HCV circulating genomes were created, by
region: the first in Japan by Prof. Masashi Mizokami et al.
(http://s2as02.genes.nig.ac.jp/); the second in the European
Union by Prof. Gilbert Deleage et al. (http://euhcvdb.fr/); and
the third in the United States by Dr. Carla Kuiken et al. (http:/
/hcv.lanl.gov/ or http://hcv-db.org). The accessibility of these
databases, which are consulted and updated by researchers
worldwide, helps standardize terms for HCV viral genotypes
and subtypes [1].
Prior to 1994, new variants were identified in Vietnam,
Thailand, Burma and Indonesia. These viral genotypes were
originally classified as genotypes 7, 8, 9, 10 and 11. These
variants were reclassified in 2005; genotype 10a came to be
denominated subtype 3k, and genotypes 7a, 7d, 7b, 7e/7c,
11a, 9a, 9b, 9c, 8b and 8a became subtypes 6e, 6c, 6d, 6f, 6g,
6h, 6i, 6j, 6k and 6l, respectively [1].
In Brazil, HCV genotype 1 is predominant, being identified
in 70% of the infected population, followed by genotypes 3
(in 25%) and 2 (in approximately 5%). In the southern region,
the profile is differentiated, the prevalence of genotype 3 being
comparable to that of genotype 1 [2,3].
The HCV viral genotype can be determined in a clinical
sample in different forms, and the regions of HCV genome
considered appropriate include the core, E1, NS4 and NS5
regions, as well as the 5’UTR, as mentioned in innumerable
studies [4,5].
The most direct method, considered the gold standard, is
the sequencing of the HCV genome in a certain region,
sufficiently divergent for different genotypes and subtypes
to be distinguished [5].
2007;11 (5) Suppl. 1:25-27.
In practice, there are two categories of assays for the
diagnosis of HCV viral types: serotyping and genotyping [6,7].
The enzyme-linked immunosorbent assay, also known as
the indirect method (serotyping), commercialized only for
research, discriminates among the six HCV genotypes, and
not the subtypes. It presents genotype-specific antibodies
for different HCV genotypes (Serotype HCV 5’NC – AbbottMurex) in the NS4 region of the genome. This test omits the
determination of the quantity of circulating viral particles and
presents lower sensitivity and specificity in the diagnosis of
HCV genotypes, when compared with genotyping. Its
performance is also limited in the evaluation of
immunocompromised patients [3,7,8].
Genotyping (the direct method) analyzes the sequences
of various regions of the genome in the differentiation of HCV
genotypes and subtypes. Therefore, it depends on samples
that present viral particles for analysis. These techniques are:
sequencing, hybridization with genotype-specific probes, and
real-time polymerase chain reaction (PCR) [5,7,9].
Reverse hybridization, distributed under the commercial
name Line Probe Assay (LIPA - Versant HCV Genotype assay
1.0/2.0; Innogenetics, Ghent, Belgium; distributed by Siemens
Medical Solutions Diagnostics, Tarrytown, NY, USA), as
described by Lieven Stuyver et al. in 1993, uses nitrocellulose
tapes, on which oligonucleotide probes of region 5’NC,
complementary to each specific HCV genotype/subtype, are
immobilized. These tapes are hybridized under high stringency
conditions, and the PCR product is marked with biotinylated
primers in the amplification process. After hybridization, an
avidin conjugate will bind to the biotinylated hybrid. The
substrate will yield the formation of a colored product, which
will be deposited on the tape and reveal the viral genotype
present in the sample. Genotypes 1 through 6, as well as
subtypes 1a, 1b, 1a/1b, 2a/2c, 2b, 3a-c, 4a-h, 5a, 6a and 10a, are
discriminated. The results are evaluated through visual
assessment.
A new version of LIPA (version 2.0) adds probes of the
core region of HCV and enables distinction of subtypes 1c of
6 and subtypes 1a and 1b, which, in the former version, was
not possible for all the samples analyzed, since region 5’NC is
extremely conserved and may not present sufficient diversity
for the distinction of these subtypes. The interpretation of
results is also performed by assessing the nitrocellulose tapes
with a scanner, and the images are analyzed using a computer
program. The new version is in the registration phase for
distribution in Brazil [7,10,11].
Direct sequencing is the most complete information on
the variations of the sequences analyzed. In addition, it is an
ideal and definitive method for the study of viral genetic
variability. Moreover, the study of viral dynamics in the context
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26
of quasispecies, during the natural history of the disease or
as consequence of antiviral therapy, depends principally on
the direct sequencing of certain regions of the HCV genome.
Furthermore, it is considered the gold standard for determining
HCV genotypes and subtypes [7].
The sequencing technique for HCV genotyping consists
of PCR amplification of part of the viral genome, especially of
the 5’NC, NS5B and core regions. These regions are
sufficiently conserved for the development of reliable primers
and, at the same time, present diversity for the discrimination
of viral genotypes and subtypes. The PCR employed for
sequencing comes from PCR-HCV qualitative or
quantitative PCR HCV products, the volume of DNA and
the high purity of the resulting tapes being essential for
the success of the analyses. This second PCR is performed
specifically to adjust or mark these products for reading in
sequencers. The assessment of both tapes, positive and
negative, is essential in order to resolve possible
ambiguities during the evaluation of the sequencings [7].
The sequences provided by the equipment should be
analyzed, and there are international databases available
for consultation, for this purpose. These databases are also
used for the centralization and collection of sequences
worldwide. The database recommended for Brazil is the
European site http://euhcvdb.fr/, probably because of the
epidemiological pathway followed during the Brazilian
colonization period.
The genotyping test TRUGENE-SIEMENS HCV 5’NC
Genotyping Kit (Siemens Medical Solutions Diagnostics,
Tarrytown, NY, USA) offers software used in conjunction
with the equipment, with a previously selected genomic
library that analyzes the sequences of region 5’NC
immediately after sequencing. In addition to the genotyping
results, it presents the homology with patterns of HCV
genotypes and subtypes based on region 5’NC. This
methodology is only available to researchers [1,7,12].
The real time PCR-HCV method is a quantitative
molecular diagnostic test that uses TaqMan technology.
In this test, genotype-specific probes marked for HCV
typing were employed in order to identify genotypes 1, 2
and 3a. Another test, also based on TaqMan technology,
can identify genotypes 1 to 4 and presents specific probes
for subtypes 1a/b, 2a/b/c, 3a and 4b/c/d. Both
methodologies are based on region 5’NC [9,13-16].
Commercially, but only for research, there is the Abbott
Real-Time PCR HCV Assay (Abbott Diagnostics Europe,
Wiesbaden, Germany) test, which also determines the viral
genotype of the samples for HCV genotypes 1a, 1b, 2a, 2b, 3,
4, 5 and 6. This methodology for the genotyping of HCV is
associated with viral quantification, levels below 6053 IU/mL
compromising the efficiency and sensitivity of the test [9].
The identification of genotypes is clinically important, and
treatment protocols recommend that genotype information
be obtained in order to delineate the duration and type of the
medication to be used. The literature mentions that genotypes
1 and 4 are considered more resistant than are genotypes 2
and 3, and that the standard treatment consists of interferon
combined with ribavirin. Another disadvantage of genotypes
1 and 4 is that they present a worse prognosis of evolution of
the disease. For treatment-naïve patients, infection with
genotype 1, 4 or 5 should be treated for 12 months, compared
with 6 months for infection with genotype 2 or 3. The
Consensus Group recommends pegylated interferon
combined with ribavirin for genotype 1. For re-treatment,
pegylated interferon associated with ribavirin is
recommended, regardless of the genotype, in the usual doses,
respecting the duration recommended for genotype 1 for other
genotypes. In cases of HIV/HCV co-infection, the response
to antiviral therapy for HCV is not favorable: 14%-38% for
genotype 1; and 43%-73% for genotypes 2 and 3 [1,7,17,18].
The advantages of genotyping methods include reliability
and the opportunity to obtain important information on the
molecular pathogenesis of HCV [7].
The impact of HCV heterogeneity and its different
genotypes on the everyday clinical management of HCV
chronic infection has not been completely established, nor
has its role as an epidemiological marker been clarified.
The sensitivity and specificity of serological and virological
tests can also be influenced by the heterogeneity of HCV,
which justifies constant evolution in the study of patients
and differentiation techniques of HCV genotypes and
subtypes [4].
References
1. Simmonds P., Bukh J., Combet C., et al. Consensus proposals for
a unified system of nomenclature of hepatitis C virus genotypes.
Hepatology 2005;42:962-73.
2. Campiotto S., Pinho J.R.R., Carrilho F.J., et al. Geographic
distribution of hepatitis C virus genotypes in Brazil. Brazilian
Journal of Medical and Biological Research 2005;38:41-9.
3. Cavalheiro N.P., Barone A.A., Tengan F.M. HCV Serotypes in
brazilian patients. Int J Infect Dis 2002;6:228-32.
4. Pawlotsky J.-M. Diagnostic tests for hepatitis C. J Hepatol
1999;31 Suppl. 1:71-9,.
5. Laperche S., Lunel F., Izopet J., et al. Comparison of Hepatitis C
Virus NS5b and 5_ Noncoding Gene Sequencing Methods in a
Multicenter Study. Journal of Clinical Microbiology 2005:733-9.
6. Pawlotsky J.-M., Prescott L., Simmonds P., et al. Serological
determination of hepatitis C virus genotype: Comparison with
a standardized genotyping assay. J Clin Microbiol
1997;35:1734-9.
7. Stéphane C., Pawlotsky J.-M. Hepatitis C virus: Virology, diagnosis
and management of antiviral therapy. World J Gastroenterol
2007;13(17):2461-6.
8. Prescott L.E., Simmonds P. Serological genotyping using synthetic
peptides derived from the NS4 region. In: LAU, JOHNSON
YIU-NAM. Hepatitis C protocols: methods in molecular
medicine. Totowa, Humana Press, cap.17, p.199-205, 1998.
9. Raymond H.W., Carol Cimmins. Evaluation of the Abbott
Molecular Diagnostics Real Time PCR Assay for HCV
Quantitative Viral Load and HCV Genotyping. Poster S30,
Clinical Virology Symposium, 2004.
10. Nadarajah R., Khan Y., Miller S.A., Brooks G.F. Evolution of a
new generation Line-Probe Assay that detects 5’untranslated
and Core regions to genotype and subtype Hepatitis C Virus. Am
J Clin Pathol 2007;128:300-4.
www.bjid.com.br
11. Bouchardeau F., Cantaloube J.F., Chevaliez S., et al. Improvement
of Hepatitis C Virus (HCV) Genotype Determination with the
New Version of the INNO-LiPA HCV Assay. Journal of Clinical
Microbiology 2007;45:1140-5.
12. Nolte F.S., Green A.M., Fiebelkorn K.R., et al. Clinical evaluation
of two methods for genotyping Hepatitis C virus based on
analysis of the 5’noncoding region. Journal of Clinical
Microbiology 2003;41:1558-64.
13. Lindh M., Hannoun C. Genotyping of hepatitis C virus by Taqman
real-time PCR. Journal of Clinical Virology 2005;34:108-14.
14. Moghaddam A., Reinton N., Dalgard O. A rapid real-time PCR
assay for determination virus genotypes 1, 2 and 3a. Journal of
Viral Hepatitis 2006;13:222-9.
27
15. Rolfe K.J., Alexander G.J.M., Tim G.W., et al. A real-time Taqman
method for hepatitis C virus genotyping. Journal of Clinical
Virology 2005;34:115-21.
16. Mukaide M., Tanaka Y., Kakuda H., et al. New combination test
for hepatitis C virus genotype and viral load determination
using Amplicor GT HCV MONITOR test v2.0. World J
Gastroenterol 2005;11(4):469-75.
17. Poynard T., Marcellin P., Lee S.S., et al. Randomized trial of interferon
alfa 2b plus ribavirin for 48 weeks or for 24 weeks versus interferon
alfa 2bplus placebo for 48 weeks for treatment of chronic infectious
hepatitis C virus. Lancet 1998;352:1426-32.
18. Tom Wong, Samuel S.L. Hepatitis C: a review for primary care
physicians. CMAJ 2006;174(5):649-59.
www.bjid.com.br
28
Noninvasive Means of Diagnosing Liver Fibrosis in Hepatitis C
Eduardo Sellan Lopes Gonçales, Adriana Flávia Feltrim Angerami and Fernando Lopes Gonçales Junior
Study Group on Hepatitis, Infectious Diseases Division, UNICAMP; Campinas,SP
Liver biopsy is still considered the gold standard for
staging fibrosis in chronic liver diseases. However, liver
biopsy is an invasive procedure, and complications occur in
0.6%-5% of patients [1,2]. In addition, to perform the procedure
there is a need for additional resources such as
ultrasonography. Therefore, as a rule, patients undergoing
liver biopsy are hospitalized for at least 6 hours [3].
Recent studies involving patients with chronic hepatitis
C showed that fragments of technically inadequate hepatic
tissue frequently lead to the underestimation of the stage of
liver fibrosis [4]. That rate of diagnostic error can vary from
10%-30% depending on the study [5]. In addition, in
developed countries, there is greater patient resistance to
undergoing biopsy.
In Brazil there is an additional factor, which is that patients
are obligated to submit to liver biopsy for indication of
treatment, except in clinically confirmed cases of hepatic
cirrhosis according to the Ministry of Health guidelines.
For all of these reasons, an increasing number of studies
are being conducted in order to evaluate the effectiveness of
noninvasive markers for staging liver fibrosis. The
noninvasive methods used in the largest number of published
studies are the calculation of two indices - the aspartate
aminotransferase (AST) to platelet ratio index (APRI) and
the FibroTest index - and the FibroScan test.
The effectiveness of the various methods evaluated in
various studies revealed quite heterogeneous results. The
APRI method and the Forns index are unable to stage a large
percentage of patients, and their accuracies do not exceed
80-85%. Therefore, a considerable number of patients are
required to undergo liver biopsy. Otherwise, approximately
20% would be incorrectly diagnosed. The efficacy of those
methods encounters difficulty regarding standardization and
the definition of cut-off values for each degree of fibrosis.
cut-off values are used. The absence of cirrhosis (Ishak stage
0-4) is defined as values lower than 1, and cirrhosis (Ishak
stage 5-6) is defined as values higher than 2 [6]. The formula
for calculating the APRI test is as follows:
APRI
The APRI was developed by Wai et al. [6] and is calculated
based on AST levels and platelet counts. According to the
results obtained in that study, the lower and upper cut-off
values for the definition of significant fibrosis and cirrhosis
are determined. Through analysis of the results, the positive
and negative predictive values for the presence or absence
of significant fibrosis or cirrhosis are also determined.
To evaluate significant fibrosis, the following cut-off
values are used: lower than 0.5 (absence of significant fibrosis,
Ishak stage 0-2); and higher than 1.5 (presence of significant
fibrosis, Ishak stage 3-6)[6]. To evaluate cirrhosis, different
FibroScan - A New Noninvasive Method
The evaluation of the degree of liver fibrosis is of
fundamental importance to the prognosis, follow-up and
therapeutic decision-making for patients with chronic liver
disease. Biopsy is an invasive method and occasionally
(although rarely) results in complications. In addition, the
biopsy results, from an anatomical-pathological point of view,
are often evaluated subjectively [8,9]. Nevertheless, biopsy
continues to be the gold standard by which fibrosis is staged
and evaluated
There are various studies on noninvasive options in the
staging, evaluation and monitoring of liver fibrosis. FibroScan
is a new method, still only available on a small scale, which
presents better results in various studies with respect to
differentiating between cirrhotic and noncirrhotic patients. It
2007;11 (5) Suppl. 1:28-31.
APRI = AST(/LSN) × 100 / Platelets (109/L)
Table 1 shows the APRI values obtained.
Table 1. The aspartate aminotransferase to platelet ratio index
results obtained by Wai et al.
No Fibrosis
Fibrosis
No Cirrhosis
Cirrhosis
Cut-Off
< 0.5
> 1.5
< 1.0
> 2.0
PPV
64%
91%
35%
65%
NPV
90%
65%
100%
95%
PPV=positive predictive value; NPV=negative predictive value.
Therefore, the aforementioned Wai et al. showed that the
APRI has a high positive predictive value to identify patients
with significant fibrosis and a high negative predictive value
to rule out cirrhosis. That study also showed that it is possible
to predict the presence or absence of significant fibrosis in
51% of patients and to predict the presence or absence of
cirrhosis in 81% of patients [6].
FibroTest
FibroTest combines and analyzes the serum levels of five
factors in patients with chronic hepatitis C. Those five factors
are bilirubin, gamma-glutamyl transferase, apolipoprotein A1,
alpha-2-macroglobulin and haptoglobin. The results obtained
are evaluated through a formula which predicts and classifies
them as F0-1, F2-3 and F4 [7].
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Noninvasive Diagnosis of Fibrosis in Hepatitis C
29
Figure 1. Algorithm proposed by Sebastiani et al. (EASL) [14], in which the aspartate aminotransferase to platelet ratio index
(APRI) method is used in conjunction with FibroTest. ALT=alanine aminotransferase; UDE=upper digestive tract endoscopy;
US=ultrasound.
Chronic hepatitis C with elevated ALT levels
APRI
F0-F1
F≥
≥2
Unclassified
FibroTest
F≥
≥2
F0-F1
Biopsy
≥F2)
Significant fibrosis (≥
F0-F1
is considered a quick and easy noninvasive procedure for
diagnosing cirrhosis and has been presented as an alternative
to liver biopsy in patients with a formal contraindication [8].
FibroScan measures hepatic elasticity through a transducer
positioned intercostally on the skin over the right lobe of the
liver. The transducer transmits low amplitude and low
frequency vibration pulses to the hepatic tissue. This
vibration pulses propagate an elastic wave whose velocity is
directly related to the elasticity of the tissue. Results are
given in kilopascals (kPa).
Some studies have shown that body mass index and age
of the patient, as well as the level of experience on the part of
the health professional, can influence the FibroScan results
of patients with chronic hepatitis C. The body mass index
and steatosis can affect the evaluation of fibrosis, although
some studies have shown that those factors are minimized
if the test is repeated a fair number of times. Some studies
recommend a total of five measurements to validate the
results [10].
For the detection of fibrosis ≥ F2, FibroScan presents
85.2% sensitivity, 90.7% specificity, 93.8% positive
predictive value, 78.8% negative predictive value and 87.7%
diagnostic power. For the detection of cirrhosis, the test
presents 78.3% sensitivity, 98.2% specificity, 97.8% positive
predictive value, 81.6% negative predictive value and 88.2%
diagnostic power [11-13].
Despite the reasonable quantity of published studies, few
have compared the methods in a randomized manner. There
are two studies that propose algorithms for evaluation. The
first study, published by the European Association for the
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30
Figure 2. Algorithm proposed by Castéra et al. [15], in which the FibroTest results are evaluated together with the FibroScan
results.
Detectable HCV
RNA
FibroScan/
FibroTest
Concordance
between the two
methods
No
Yes
Liver biopsy
No liver biopsy
Treatment or
monitoring
Fibrosis absent or
minimal (<F2)
Moderate fibrosis
(F2)
Severe fibrosis or
≥F3)
cirrhosis (≥
Monitoring
Treatment
Treatment + UDE
and US every 6
months
Study of the Liver (EASL), aims to standardize and compare
the various methods based on serum markers of liver fibrosis.
In its conclusion, the study proposes the use of an
organogram that could reduce the need for liver biopsy by
60%-70% [14].
That study, conducted by Sebastiani et al. [14], evaluated
the capacity of different methods to diagnose significant
fibrosis (METAVIR fibrosis score ≥ 2) in patients with normal
or high levels of transaminase and to diagnose cirrhosis.
The results obtained show that significant fibrosis can
be diagnosed with an accuracy of 94% using the APRI as the
first screening test, followed by FibroTest in patients who
were not classified through the APRI method, thereby limiting
biopsy to only those patients in whom the degree of fibrosis
is classified as F0-F1 using noninvasive methods [14].
Cirrhosis can also be diagnosed through this algorithm
(95% accuracy). The authors considered that in their original
study they might have obtained highly favorable results due
www.bjid.com.br
The study conducted by Castéra et al. proposes the
combination of FibroScan and FibroTest as a screening
method for significant fibrosis. When there was concordance
between the methods, which occurred in 70%-80% of patients,
the compatibility with the liver biopsy was 84% in diagnosing
fibrosis ≥ F2, 95% in diagnosing fibrosis ≥ F3, and a 94% in
diagnosing cirrhosis (F4).
Therefore, much still needs to be studied in relation to
the noninvasive methods for estimating the degree of
fibrosis, which is why it is necessary to carry out randomized
comparative studies involving different patient populations.
Table 2. FibroScan results
Variable
Sensitivity
Specificity
PPV
NPV
≥F2
67%
89%
95%
48%
Degree of fibrosis
≥F3
73%
91%
87%
81%
F4
87%
91%
77%
95%
PPV=positive predictive value; NPV=negative predictive value.
to the fact that the majority of patients presented significant
fibrosis. The principal limitation of these markers is the
difficulty in obtaining confirmation for patients with F0-F1
fibrosis. Therefore, an algorithm for the evaluation of fibrosis
that attempts to define which patients are not required to
undergo liver biopsy was developed.
Another study conducted by Castéra et al. [15] compared
the effectiveness of transitory hepatic elastography
(FibroScan, Echosens, Paris, France) in relation to the APRI
and FibroTest. It succeeded in showing that FibroScan has a
great capacity to diagnose significant fibrosis (≥ F2). The
results can be seen in Table 2.
Therefore, it can be seen that FibroScan presents a high
positive predictive value for patients with fibrosis ≥ F2 and
an excellent negative predictive value for patients with hepatic
cirrhosis. It is undoubtedly a good method for demonstrating
significant fibrosis or for ruling out hepatic cirrhosis.
31
References
1. Perrault J., McGill D.B., Ott B.J., Taylor W.F. Liver biopsy:
complications in 1000 inpatients. Gastroenterology
1978;74:103-6.
2. Gunneson T.J., Menon K.V., Wiesner R.H., et al. Ultrasoundassisted percutaneous liver biopsy perfomed by a physician
assistant. Am J Gastroenterol 2002;97:1472-5.
3. Wong J.B., Koff R.S. Watchful waiting with periodic liver biopsy
versus immediate empirical therapy for histologically mild
chronic hepatitis C. A cost-effectiveness analysis. Ann Intern
Med 2000;133:665-75.
4. Colloredo G., Guido M., Sonzogni A., Leandro G. Impact of liver
biopsy size on histological evaluation of chronic viral hepatitis:
the smaller the sample, the milder the disease. J Hepatol
2003;39:239-44.
5. Poniachik J., Bernstein D.E., Reddy K.R., et al. The role of
laparoscopy in the diagnosis of cirrhosis. Gastrointest Endosc
1996;43:568-71.
6. Wai C.T., Greenson J.K., Fontana R.J., et al. A simple
noninvasive índex can predict both significant fibrosis and
cirrhosis in patients with chronic hepatitis C. Hepatology
2003;38:518-26.
7. Poynard T., McHutchison J., Manns M., et al. Biochemical
surrogate markers of liver fibrosis and activity in a randomized
trial of peginterferon alfa-2b and ribavirin. Hepatology
2003;38:481-92.
8. Transient elastography: a new surrogate marker of liver fibrosis
influenced by major changes of transaminases. Journal of Viral
Hepatitis 2007;14:360-9.
9. Diagnosis of hepatic steatosis and fibrosis by transient
elastography in asymptomatic healthy individuals: a
prospective study of living related potencial liver donors.
Journal of Gastroenterol 2007;42:382-8.
10. Usefulneness of elastometry in evaluating the extents of liver
fibrosis in hemophiliacs coinfected with hepatitis C and
human immunodeficiency virus. Hepatology research
2006;35:135-9.
11. Features associated with success rate and performance of fibroscan
measurements for the diagnosis of cirrhosis in HCV patients: a
prospective study of 935 patients. Journal of Hepatology
2007;46:628-34.
12. Transient elastography: a valid alternative to biopsy in patients
with chronic liver disease. Journal compilation 2006;24:513-8.
13. Do not trivialize the Fibroscan examination, value its accuracy.
Journal of Hepatology 2007;46.
14. Sebastiani G., Vario A., Guido M., et al. Stepwise combination
algorithms of non-invasive markers to diagnose significant
fibrosis in chronic hepatitis C. Journal of Hepatology
2006;44:686-93.
15. Castéra L., Vergniol J., Foucher J., et al. Gastroenterology
2005;128:343-50.
www.bjid.com.br
32
Chronic Hepatitis C: Pathological Anatomy
Evandro Sobroza de Mello and Venâncio Avancini Ferreira Alves
Pathological Anatomy Division, Hospital das Clínicas of São Paulo; LIM-14: Hepatic Pathology,
University of São Paulo School of Medicine; CICAP – Hospital Alemão Oswaldo Cruz; São Paulo, SP, Brazil
In infections with the hepatitis C virus (HCV), there is a
wide spectrum of histological alterations that can affect the
liver, from acute hepatitis to mild reactive phenomena to more
severe forms, including chronic hepatitis with varying degrees
of inflammation/fibrosis, cirrhosis, and hepatocellular
carcinoma. In cases of acute hepatitis C, biopsies are rare,
pathologists focusing their attention on the chronic form of
the disease. The histological diagnosis of chronic hepatitis
through liver biopsy remains extremely important in the
management of patients infected with HCV, since it is the
cornerstone of the detection of liver disease caused by the
virus as well as the determination of the intensity of this
disease. It should therefore be added to the diagnosis of
infection made using serologic methods.
The basic parameter for the histological diagnosis of
chronic hepatitis is the presence of portal inflammatory
infiltrate, with predominance of lymphocytes, usually with
variations in the number of plasmocytes and histiocytes. This
inflammation is accompanied by periportal activity of varying
degrees (also denominated interface activity or piecemeal
necrosis), parenchymal activity (lobular) and fibrosis.
There are various classification systems using in the
scoring and staging of chronic hepatitis [4,9,11,13,18,21,34].
Many of those systems are of historical importance. According
to directive no. 863, issued by the São Paulo State Secretary
of Health on November 4, 2002, it is recommended that one of
two chronic hepatitis classification systems be used: the
Sociedade Brasileira de Patologia (SBP, Brazilian Society of
Pathology) system [13] or the METAVIR system [1,4]. These
two systems are in fact very similar, and they both take into
account the previously mentioned basic aspects of chronic
hepatitis: periportal activity, lobular activity, and fibrosis. In
addition to these, the classification system proposed by Ishak
in 1995 [18] has been widely used in international literature.
The Ishak system is an update of the system proposed by the
same author in 1981, which gained popularity and was
commonly referred to as the Knodell system [21] (a designation
that should no longer be used), has been widely used in
international literature. Table 1 shows an approximate
correspondence between these systems, both for fibrosis
(architectural alteration) and for periportal/lobular activity.
Protocol of Histological Evaluation for Liver Biopsies of
Patients with Chronic viral Hepatitis
This protocol can be applied to several etiologies of
chronic hepatitis, including, in addition to HCV, HBV, autoThe Brazilian Journal of Infectious Diseases
2007;11 (5) Suppl. 1:32-36.
immune hepatitis and, less frequently, Wilson’s disease or
some forms of drug-induced hepatitis. The protocol is based
on the criteria of the SBP National Consensus of Chronic
Hepatitis [13].
1) Sample type (needle biopsy, wedge biopsy, resected
surgical sample, other):
2) Sample size
Number of portal spaces in the biopsy: _____
3) Histological variables:
• Portal fibrosis:
( ) 0 (absent)
( ) 1 (discrete, without septum formation)
( ) 2 (with portal-portal septa)
( ) 3 (with portal-portal and portal-central septa, with
formation of nodules – in ‘nodular transformation’)
( ) 4 (cirrhosis)
• Portal inflammation
( ) 0 (absent)
( ) 1 (discrete)
( ) 2 (moderate)
( ) 3 (pronounced)
( ) 4 (very pronounced)
• Periportal activity (interface activity)
( ) 0 (absent)
( ) 1 (presence of spillover only)
( ) 2 (discrete piecemeal necrosis – occasional foci in
some portal spaces)
( ) 3 (moderate piecemeal necrosis – occasional foci in
many portal spaces or innumerable foci in few portal
spaces)
( ) 4 (pronounced piecemeal necrosis - innumerable foci
in many portal spaces)
• Parenchymal activity
( ) 0 (absent)
( ) 1 (tumefaction, lymphocyte sinusoidal infiltrate and
occasional foci of lytic hepatocytic necrosis)
( ) 2 (innumerable foci of lytic hepatocytic necrosis)
( ) 3 (occasional areas of confluent necrosis)
( ) 4 (innumerable areas of confluent necrosis or areas
of panacinar necrosis)
• Histological evidence of association with other conditions:
( ) level ______ siderosis
( ) steatohepatitis markers
( ) others:______
Nature and Size of the Liver Biopsy
Surgical biopsies performed with forceps generate
subcapsular samples and should be discouraged, since the
portal spaces in this location are frequently large, and it is
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Chronic Hepatitis C – Pathological Anatomy
33
Table 1. Approximate equivalence of the most widely used classification systems for the staging and scoring of chronic hepatitis
Architectural Alteration (Fibrosis)*
SBP, 2000
METAVIR, 1994
Ishak, 1995
0
1
2
3
4
0
1
2
3
4
0
1 or 2
3
4 or 5
6
Inflammatory Activity**
SBP, 2000 and Ishak, 1995
Parenchymal activity
0 or 1
0 or 1
2
2
2
3
3
4
METAVIR, 1994
Periportal activity
A
0
1 or 2
0–1
2
3–4
0–2
3–4
0–4
0
1
1
2
3
2
3
3
SBP= Sociedade Brasileira de Patologia (SBP, Brazilian Society of Pathology). *Maximum Ishak
score, 6; maximum METAVIR score, 4; maximum SBP score, 4. **Corresponds to periportal and
parenchymal activity, independently, for SBP and Ishak, and mixed periportal and lobular for METAVIR;
in the METAVIR classification, the activity score reaches 3, whereas in Ishak and SBP it reaches 4.
difficult or impossible to correctly evaluate the presence of
fibrosis. Even during the surgical procedure, therefore, liver
biopsy should be performed with a needle. In addition, the
biopsy should preferably be performed at the outset of the
surgery in order to prevent alterations secondary to surgical
manipulation.
Data in the literature demonstrate that the size of the needle
biopsy greatly influences the result of the analysis
[8,10,15,33,35]. Samples measuring 3.0 cm or more in length,
show hepatitis with mild activity, as a result, in only 50% of
the cases; 1.5-cm long samples, in 60%; and those measuring
1.0 cm or less, in almost 90% of the cases [8]. Other authors
have also considered 1.5 cm as the minimum size for diagnosis
in needle liver biopsy [35]. Thin needles have also provided
inferior results [8,33]. Bedossa et al. [3] only achieved a
precision plateau with 2.5-cm long biopsies. Therefore, 1.5cm long biopsies should be considered the minimum
necessary size and, ideally, they should measure 2.5 cm or
more. Larger diameter needles, such as Tru-Cut needles, are
also recommended.
Steatosis
Approximately 50% of the biopsy samples collected from
patients with HCV present steatosis [17,40]. The evaluation
of the presence of steatosis, its scoring, and the evaluation of
the presence of associated steatohepatitis has gradually
become more important [6,7,12,16,19,20,24,29-32,36,40].
The spectrum of steatosis, steatohepatitis and cirrhosis has
been denominated nonalcoholic fatty liver disease (NAFLD).
Although NAFLD is common in the population in general,
concomitance between NAFLD and HCV is 2-3 times greater
than what would be expected only at random [24]. In patients
with chronic HCV infection, steatosis has been attributed to a
series of factors usually associated with NAFLD, including
high body mass index, insulin resistance and old age [16,26,31].
Evidence also indicates that steatosis contributes to the
progression of fibrosis in a pattern similar to that seen in NAFLD
[7,16,17,40].
It has been suggested that steatosis can also result from
the viral cytopathic effect, especially in patients infected with
genotype 3. In a series of patients with genotype 3 and steatosis,
a sustained virological response led to regression of steatosis in
91% of the cases, a much higher index than the 19% observed for
those who did not present sustained virological response [6],
making the cytopathic effect a more consistent cause of steatosis.
Other authors have reported similar results [22,36].
In HCV-positive patients, it is currently essential to
characterize steatosis and related injuries, especially the
presence and quantification of perisinusoidal and centrilobular
fibrosis, which characterizes steatohepatitis. The lesson we learn
from steatosis is that, in HCV-infected patients, biopsy is an
instrument for the detection of liver diseases, whether associated
with the virus or not, and that we should be prepared for other
(probably less common) liver diseases that might be present in
a particular case.
Histopathological Aspects of Post-Transplant HCV
Recurrence
Immediate post-transplant virological recurrence of HCV
is universal, and the progression of the disease is more rapid
www.bjid.com.br
34
than in non-transplanted patients [22A]. Long-term studies have
shown that 70-90% of cases will present histological recurrence
of the disease [20A,23A]. In most cases, hepatitis C
histologically manifests in the same manner before the
transplant: with portal and parenchymal inflammation,
aggression of the interface with piecemeal necrosis, and fibrosis
at portal spaces. Steatosis and ductal injury are also common
findings. Earlier findings are often predominantly lobular, with
inflammation and apoptotic bodies (acute hepatitis), and
steatosis is occasionally the first histological manifestation
[4A]. In a small proportion of cases, hepatitis C can result in a
severe, rapidly progressive cholestatic pattern, which leads to
loss of the graft [36A,41,42]. Due to the low tolerance of
transplanted individuals to the treatment with interferon and
ribavirin [24A,45], the evaluation of the biopsy is crucial for
indication of treatment. Some anatomopathological aspects can
be useful in predicting its evolution: histological recurrence in
less than six months [46]; level of inflammatory activity [23A,44];
marked ballooning of hepatocytes; and cholestasis [43].
Histopathological Criteria for Possible Predictive Value of
Worse Evolution
In chronic hepatitis, the contribution of the
histopathological analysis of the samples collected by liver
biopsy is currently considered decisive for diagnosis, for
staging of the architectural damage, and for determining the
level of necroinflammatory activity, assuming a decisive role
in indicating the therapy with antiviral agents.
In our view, in addition to reports on that decision, as
summarized in the METAVIR, Ishak, Scheuer, and Desmet
classification systems, or, among us, the SBP/Brazilian Society
of Hepatology consensus led by Gayotto, most recent
evidence brings back the need of a detailed report of each of
the principal forms of liver damage, and there have been
studies that demonstrate a more rapid evolution of cases that
present, among other predictive factors, more interface activity,
confluent necrosis of hepatocytes, and steatosis [47,48].
A study involving 106 patients with initial biopsy
presenting architectural staging 0 or 1 and re-biopsied after a
mean interval of 7.8 years (minimum of 48 months) [48] revealed
progression of architectural damage in 64 cases (60.4%),
suggesting the need for therapeutic intervention, even in
infected individuals not yet presenting significant alterations
to the hepatic architecture. Among the predictive factors for
progression of the injury, those authors highlight the level of
necroinflammatory activity: 31.2% of the cases with moderate
activity (A2) presented progression, which only occurred in
2.3% of those without activity (A0) and the presence of
steatosis (progression in 87.5% of the cases with > 30% cells
with steatosis, in 80% of those with < 30% steatosis, and in
only 48.6% without steatosis).
Other authors also emphasize the presence and extent of
steatosis as a risk factor for progression of injuries in chronic
hepatitis C, either resulting from viral cytopathic effect, as
proposed for genotype 3a [49], or associated with coexistent
steatohepatitis, alcoholic [50] or nonalcoholic [47]. A recent
meta-analysis including 3068 Italian patients infected with
HCV and submitted to biopsy [51] confirmed that steatosis is
independently associated with genotype 3, fibrosis, diabetes,
inflammatory liver activity, drinking, body mass index, and
older age.
Important experience was brought to debate in the most
recent European Hepatology Congress: analyzing predictive
factors of damage progression in 563 cases of HCV with mean
intervals between biopsies of 5.4 years [52], it was determined
that, in contrast to generic approaches that suggested that
liver damage progress in a relatively uniform, linear manner, the
speed of progression varied considerably in each patient. These
authors, selecting statistically significant variables, identified
the risk of progression associated with various architectural
alterations (Table 2).We find, therefore, that important current
studies demonstrate the outstanding contribution of
histopathological findings in the discrimination of differentiated
progression risk in patients chronically infected with HCV. More
than dividing patients in classes that deserve antiviral treatment
or not, most recent evidence point to the need of reviewing the
systems of histological scoring, and the pathologist should
inform, in addition to the stage of architectural alteration, the
level of each type of necroinflammatory damage in each acinar
compartment of the liver.
Table 2. Progression risks of architectural alterations
Factor
Age > 50 years
Brindging necrosis
Confluent necrosis
Piecemeal necrosis
Steatosis
Moderate/Pronounced
Discrete
Perivenular fibrosis
Relative Risk
1.6
4.0
3.4
2.9
3.8
2.1
2.6
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C virus quasispecies in patients with severe cholestatic hepatitis
after liver transplantation. Hepatology 1999;30:1513-20.
42. Troppmann C., Rossaro L., Perez R.V., McVicar J.P. Early, rapidly
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www.bjid.com.br
37
Co-Infection with Hepatitis B Virus and Hepatitis C Virus
Heloísa Pedrosa Mitre and João Silva de Mendonça
Infectious Diseases Division of the Hospital for State Civil Servants; São Paulo, SP, Brazil
The hepatitis B virus (HBV) and the hepatitis C virus (HCV)
share common transmission pathways. Therefore, co-infection
can be expected. The World Health Organization estimates
that, worldwide, 170 million people are infected with HCV, and
350 million people are infected with HBV. However, the number
of individuals co-infected with both viruses is unknown.
Although various studies have evaluated small numbers
of co-infected individuals, the inclusion criteria, parameters
assessed, and study designs are not uniform. In addition,
ethnicity, local epidemiology, and viral genotypes are also
diverse. Therefore, the conclusions of a specific study, in
principle, should not be widespread.
In cases of co-infection with HBV and HCV, the replication of
either virus can be inhibited, just as either virus can be dominant
or the dominance can alternate between the two. It is more common
for HBV to appear to be suppressed by HCV. The chronologies
of the two infections have an influence on which virus will be
dominant. Molecular biology techniques (to determine levels of
HCV RNA and HBV DNA) have facilitated the definition of the
interaction between the two viruses.
Co-infections can appear in various manners:
a) Simultaneous acute infection with HBV and HCV:
presupposes same source and transmission pathway. The
number of studies is small, but indicates that the interaction
between the two viruses is similar to that which occurs in
chronic infections. There are descriptions of cases in which
there is a delay in the identification of the Hepatitis B surface
antigen (HBsAg), lower levels of alanine aminotransferase
(ALT), and lower HBV antigenemia, which can be attributed
to suppression of HBV activity by HCV.
b) Superinfection by one virus, the other virus being
chronically present: it should be suspected above all in
individuals with risk factors, such as the use of illicit
intravenous drugs, multi-transfused individuals, and those
living in areas of high HBV prevalence.
Superinfection by HCV: infection by HCV in a patient that
is already infected with HBV. This is known in Asian countries,
where the prevalence of HBV is high.
The viral suppression of the HCV may occur, although the
viral suppression of the HBV is more frequent, noted by lower
levels of HBV DNA and lower DNA polymerase activity, as
well as by the hepatic expression of HBsAg and hepatitis B
core antigen (HBcAg), clearance of the hepatitis B e antigen
(HBeAg) or even of the HBsAg.
The HCV core antigen seems to affect the transcription of
HBV and, as a result, its replication, which is reported to be
more accentuated for the HCV genotype 1.
2007;11 (5) Suppl. 1:37-39.
After HBV clearance, HCV can persist, resulting in chronic
hepatitis. In addition, there is the possibility of evolution to
severe disease, with a risk of death.
Superinfection by HBV: infection by HBV in an individual
that is already chronically infected with HCV. The HCV RNA
levels are lower, and, in a study conducted in Italy, HCV
clearance was higher in co-infected individuals (71%) than in
mono-infected individuals (14%).
However, the HBV DNA levels can be lower than those of
mono-infected individuals, indicating HCV interference.
Therefore, one virus can induce the clearance of another.
The evolution to severe forms of the disease, with
fulminant hepatitis profiles, has also been described.
c) Asymptomatic infection with HBV: there are reports of
patients infected with HCV, with low levels of HBV DNA,
reactive anti-HBc, however, non-reactive HBsAg, HBeAg,
anti-HBe, and anti-HBs, configuring co-infection with HCV
with asymptomatic HBV.
These individuals evolve with high ALT levels and high
histological activity. There are reports in which biopsies
from such patients were evaluated, and cirrhosis was found
in 33% of the cases, compared with 19% for patients
presenting chronic mono-infections.
The data suggest that the evolution of the disease is more
severe in co-infected individuals.
There are various potential outcomes:
a) Clearance of both viruses: negative HBsAg and,
eventually, appearance of anti-HBs. The anti-HCV remains
reactive. However, both HBV DNA and HCV RNA become
undetectable. This is the best evolution for both viruses.
b) Fulminant hepatitis: several studies indicate that
individuals co-infected with HBV and HCV have a higher
risk of evolution to fulminant hepatitis. In a prospective
study involving patients with acute hepatitis C, of those
who died, 23% were chronically infected with HBV, whereas
only 2.9% were infected with HBV (OR=10.2).
In a study conducted in France, among 40 patients with
fulminant or sub-fulminant hepatitis, 12.5% presented acute
hepatitis B or C, and 7.5% presented superinfection with
HCV.
c) Chronic hepatitis: of all possible events, this is the one
that occurs most frequently and presents treatment
possibilities. Among the chronic cases of hepatitis, several
immunological and molecular-biological profiles can be
identified:
Active HBV and HCV: detectable HBV DNA and HCV RNA
in serum. This situation has higher possibility of evolution to
cirrhosis and hepatic decompensation. In these cases,
treatment may be considered, either with interferon (IFN) +
ribavirin (RBV) or IFN + lamivudine (LMV).
www.bjid.com.br
38
HBV/HCV Co-Infection
Inactive HBV and active HCV: with evolution to undetectable
HBV DNA and detectable HCV RNA. If treatment is indicated,
IFN + RBV is the treatment of choice.
Active HBV and inactive HCV or previous infection with
HCV: with reactive HBsAg and HBeAg, detectable HBV DNA
and undetectable HCV RNA.
If indicated, therapeutic options may be IFN or IFN + LMV.
Cirrhosis: co-infected individuals have a higher risk of
evolution to cirrhosis if compared with mono-infected
individuals (44% and 21%, respectively) as well as a higher
risk of chronic hepatitis decompensation.
Hepatocellular carcinoma: evidence indicates that the
possibility of evolution to hepatocellular carcinoma (HCC) is
higher in co-infected individuals. A prospective study
comprising 290 individuals with cirrhosis concluded that, in
the univariate and multivariate analyses, co-infection with
HBV and HCV is a predictive factor for the development of
HCC. These conclusions were confirmed in subsequent
studies, in which the incidence of HCC in individuals coinfected with HBV and HCV was 2 and 3.7 cases/100
individuals/year, respectively.
The cumulative risk of HCC development in 10 years was
45% in co-infected individuals, 16% in individuals monoinfected with HBV, and 28% in individuals mono-infected with
HCV.
Treatment
The treatment guidelines have been defined in monoinfected individuals, among others, by professional associations
of liver researchers, such as the Asian-Pacific Association for
the Study of the Liver, the European Association for the Study
of the Liver, and the American Association for the Study of
Liver Diseases. According to these guidelines, the choice of
pegylated interferon over conventional interferon was defined
in association with ribavirin in cases of chronic infection with
HCV, and, in isolation, in cases of chronic infection with HBV,
when the administration of antiviral drugs is also considered.
On the other hand, the treatment of individuals co-infected
with HBV and HCV is complex due to the interaction between
both viruses, and between both viruses and the host
immunologic system. Standards for these treatments are not
yet available. However, the therapeutic regimen should be
carefully chosen and should be based on serologic markers,
viremia levels, histology, and, above all, definition of the
dominant virus.
Individuals with uncompensated cirrhosis should be
referred to facilities specializing in liver transplantation.
Interferon
Since IFN is an immunomodulating drug, with antiviral
and antiproliferative effect, it is effective against HBV and
HCV. It is also the most widely studied therapeutic option.
The first study in co-infected individuals, carried out in the
1990s, suggests that higher doses (9 MU 3 times a week) are
more effective for the clearance of HBV or HCV.
If suppression of one of the viruses is achieved, there is
the possibility of reactivation of the other, since the suppressor
effect of the former has been removed. Pegylated IFN, which
is more effective in mono-infected individuals, can have a
similar effect in co-infected individuals.
In the infection with HCV and asymptomatic HBV
infection, the histological changes and activity are typically
pronounced. The response to treatment is less favorable and
respondents present recurrence (downregulation?).
IFN + RBV: this is the regimen that performs best in
individuals mono-infected with HCV. There have been various
studies, involving small samples of co-infected individuals,
reporting rates of sustained biochemical response and
sustained virologic response (SVR) that are comparable to
those of mono-infected individuals, especially if HCV is the
dominant virus.
Due to the viral interaction, it is extremely important that
HBV activity is monitored. It is known that at least half of the
patients present HBV reactivation at the beginning of the
treatment, and that 45% will have ‘flare-ups’. Therefore, we
should pay attention to HBV, even if HBV DNA is undetectable
at the beginning of the treatment. Over the course of the
disease, HBV DNA can become undetectable in 10% to 30%
of the patients.
IFN + LMV: small studies suggest that the addition of LMV
is useful, above all in patients with active HBV. One such study
described eight HBeAg-reactive patients, in whom HBV DNA
and HCV RNA were detected, submitted to a 12-month course
of IFN + LMV, followed by an additional six months of treatment
with LMV. In approximately one-third of the patients, there was
HBV and HBeAg clearance. In half of the patients, there was
normalization of ALT, HCV clearance, and an SVR.
It would be premature to venture an opinion on what is
the most appropriate regimen, considering that further studies,
involving larger patient samples, are still needed.
Adefovir and entecavir: there have been no studies involving
co-infected individuals. These might be good options in
patients with dominant HBV.
Liver transplantation: little experience with co-infected
individuals.
There are reports of higher survival compared with
individuals mono-infected with HBV, with suggestion of the
benefit of HCV suppression over HBV in immunosuppression
after transplantation.
In order to define the risks and benefits, it is necessary to
await the results of further, larger studies.
Triple Co-Infection
a) HBV, HCV, and hepatitis D virus (HDV): only patients
who are already infected with HBV can acquire HDV. The
triple infection can occur in certain geographic areas where
the prevalence of infection with HBV is high.
The evolution to severe disease is described, the desired
treatment being that that consists of high doses of IFN for a
long period and, still, with poor responses.
www.bjid.com.br
HBV/HCV Co-Infection
There are few relevant studies in the literature, and all of
those involved small study samples, which precludes the
standardization of the procedure in these cases. There are
reports of treatment of seven patients with achievement of
sustained biochemical response in two patients, and
worsening with side effects in two other patients.
b) HBV, HCV, and HIV: in these cases, the complexity of
the HBV/HCV interaction is added to the effect of the HIV on
the host immunologic system.
It is known that, in HCV/HIV and HBV/HIV co-infections,
the evolution of the hepatic disease is faster and potentially
more severe. In addition, after the introduction of the highdose anti-HIV therapy known as highly active antiretroviral
therapy, the mortality rate for chronic liver diseases has
increased.
There are few studies on the treatment of individuals coinfected with HBV, HCV, and HIV. The rate at which an SVR is
achieved has been reported to be 17% with the use of IFN,
compared with 25% for IFN + RBV.
It must be borne in mind that, due to the frequent use of
LMV in the antiretroviral therapy for HIV, HBV resistance to
LMV is high in co-infected individuals.
In one small study, the effect against the dominant virus,
HBV or HCV, did not lead to the reactivation of the other,
except in one case involving a patient with a very low CD4
lymphocyte count.
39
Studies of new antiviral drugs that are more effective
against HBV (tenofovir, entecavir, adefovir, etc.) in HBV/
HIV or HBV/HCV/HIV co-infected individuals might inform
therapeutic decisions regarding the complex treatment of
co-infections involving HBV.
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8. Soriano V., et al. Treatment of chronic hepatitis B or C in HIVinfected patients with dual viral hepatitis. JID 2007;195:1181-3.
9. Wang Y.M., et al. Suppression of hepatitis C virus by hepatitis B
virus in coinfected patients at the National University Hospital
of Singapore. J Gastroenterol 1999;34:481-5.
www.bjid.com.br
40
Co-Infection with Hepatitis C Virus and Human T Lymphocyte Virus
Carlos Brites Alves
Federal University of Bahia, School of Medicine; Salvador, BA, Brazil
Most individuals infected with human T lymphocyte virus
(HTLV) type 1 or 2 will not develop the disease related to this
virus, remaining asymptomatic for the rest of their lives. This
fact has important implications for prospective counseling
and evaluation of this population. Individuals infected with this
virus, once identified, should be submitted to anamnesis and
complete physical examination (in order to identify early
manifestations of the disease and probable forms of acquiring
the infection) and should be periodically evaluated every 6-12
months. It is recommended that injection drug users be tested for
other pathogens common to this population, such as the hepatitis
B virus, the hepatitis C virus (HCV), HIV, etc. It is recommended
that partners of sexually active individuals be tested for HTLV.
Children of women infected with HTLV-1 should be tested.
Follow-up evaluations should include the following
periodic laboratory tests (every 6-12 months): complete blood
workups with platelet counts; parasitological stool
examination (testing for Strongyloides); urine and urine
sediment test (urinary infection). Although the proviral load
of HTLV-1 is still under evaluation and has yet to be validated,
some studies recommend annual quantification.
Special Situations
• In asymptomatic cases that present evidence of HTLVrelated systemic disease, such as dermatological
alterations, hyperreflexia, clone or Babinski sign:
• Serum calcium level, immunophenotyping of T
lymphocytes (CD3, CD4, CD8, histocompatibility
leukocyte antigen-DR, CD38 and CD56); diffuse
histiocytic lymphoma, creatine phosphokinase, folate
and vitamin B12 levels; free thyroxine 4 and thyroid
stimulating hormone; and study of somatosensitive
evoked potentials.
Healthy individuals infected with HTLV-1 should be
counseled regarding the transmission mechanisms of the
infection and be reassured that the probability of developing
the disease in the future is low. If necessary, they should be
referred for specialized psychological follow-up evaluation.
Currently, there is no indication – based on scientific
evidence – that any certain type of specific anti-HTLV-1
pharmacological intervention plays a role in the prophylaxis
of HTLV-related diseases. Therefore, there is no indication for
the use of immunomodulatory, immunosuppressant or
antiretroviral drugs in asymptomatic individuals infected with
HTLV (National Ministry of Health, guide of clinical
management of the HTLV-infected patient, 2004).
2007;11 (5) Suppl. 1:40-41.
HTLV/HCV Co-Infection
Infection with HCV is frequently detected in HTLV-infected
individuals and vice versa, as seen in other commonly
transmitted pathogens.
Co-infection with HTLV-2 and HCV in patients who are
drug users has been reported, principally in cohorts in the
northern hemisphere [1]. A study carried out in Paraná, Brazil
revealed a strong association (OR=22.60; 95% CI: 10.35-49.35)
between these two pathogens, probably reflecting shared
transmission forms [2].
The prevalence of co-infection also seems to increase in
individuals infected with HIV in Brazil. Segurado et al. [3]
demonstrated that HCV infection was an independent risk
factor for HTLV infection (adjusted OR=6.43, p=0.02).
Interactions Between HCV and HTLV in Co-Infected
Individuals: Potential Clinical Implications
There are few studies on the effects of co-infection with HCV
and HTLV. Hisada et al. [4] demonstrated that the co-infection
with HTLV is associated with greater viral load of HCV.
In a study conducted in Japan, HCV/HTLV-1 co-infected
individuals were found to be at a higher risk of incidental liver
disease (RR = 5.9), hepatocarcinoma and death (RR = 21.9), as
well as for developing diabetes [5]. In addition, co-infected
individuals have been shown to present a higher frequency
of anergy to purified protein derivative, although with no
statistical significance, suggesting a differentiated
immunomodulatory effect in this population [6].
A recent study conducted in the state of Bahia, Brazil
revealed a high prevalence of HTLV/HCV co-infection.
Although it did not evaluate the clinical impact, it showed the
relevance of this association.
In practical terms, there is no established recommendation
for the management of HCV/HTLV co-infection. Nevertheless,
the analysis what evidence there is suggests that this
association can result in significant modifications in the natural
history of HCV, increasing the viral load of HCV, as well as
increasing the morbidity and mortality associated with this
infection. Therefore, the co-infected patient requires special
attention regarding the clinical evolution of hepatitis C and
the markers of the infection.
In addition to monitoring the parameters related to HTLV
infection, especially the neurologic alterations secondary to
the infection, we should carefully evaluate the stage of the
liver disease, and routinely evaluate the viral load of HCV.
Attention should be given to potential alterations of glucose
metabolism, since there seems to be an increased tendency
toward these problems in the co-infected individual. For coinfected patients, routine evaluations of fasting glycemia, as
well as glucose tolerance tests, could be necessary.
www.bjid.com.br
HCV/HTLV Co-Infection
References
1. Zunt J.R., Tapia K., Thiede H., et al. HTLV-2 infection in injection
drug users in King County, Washington, Scand J Infect Dis
2006;38(8):654-63.
2. Morimoto H.K., Caterino-De-Araujo A., Morimoto A.A., et al.
Seroprevalence and risk factors for human T cell lymphotropic
virus type 1 and 2 infection in human immunodeficiency virusinfected patients attending AIDS referral center health units in
Londrina and other communities in Paraná, Brazil (AIDS Res
Hum Retroviruses 2005;21(4):256-62.
3. Segurado A.C., Braga P., Etzel A., Cardoso M.R. Hepatitis C virus
coinfection in a cohort of HIV-infected individuals from Santos,
Brazil: seroprevalence and associated factors. AIDS Patient Care
STDS 2004;18(3):135-43.
41
4. H i s a d a M . , C h a t t e r j e e N . , Z h a n g M . , e t a l . I n c r e a s e d
hepatitis C virus load among injection drug users infected
with human immunodeficiency virus and human T
lymphotropic
virus
type
II.
J
Infect
Dis
2003;188(6):891-7.
5. Boschi-Pinto C., Stuver S., Okayama A., et al. A follow-up
study of morbidity and mortality associated with hepatitis
C virus infection and its interaction with human T
lymphotropic virus type I in Miyazaki, Japan. J Infect Dis
2000;182(1):379-80.
6. Hisada M., Shima T., Okayama A., et al. Suppression of skin
reactivity to purified protein derivative by hepatitis C virus
among HTLV-1 carriers in Japan.J Acquir Immune Defic Syndr
Hum Retrovirol 1998;19(4):421-5.
www.bjid.com.br
42
Basic Guidelines for the Treatment of HIV/HVC Co-Infection
Edgard De Bortholi
The treatment of HIV/HCV co-infection presents many
questions that have not yet been answered or on which there
is no consensus. Since it is a recently introduced issue, the
guidelines in the literature are divergent on some points. The
still fragmented knowledge and lack of long-term worldwide
experience in the treatment of such co-infections has forced
referral facilities to constantly update their approaches.
Some guidelines for the management of the HIV/HCV coinfected patient have been proposed, and new
recommendations are particularly necessary:
• Management of patients with persistently normal
aminotransferase levels.
• Definition and quantification of liver fibrosis: when
and how?
• Predictors of the response to anti-HCV therapy in coinfected patients.
• Therapeutic doses of pegylated interferon and ribavirin.
• Treatment duration.
• Treatment of nonresponsive and recidivist patients.
• Treatment of acute infection in HIV-positive patients.
• HIV/HCV/HBV co-infected patients.
• Interaction between antiretroviral drugs and anti-HCV
therapeutics.
• Antiretroviral hepatotoxicity in co-infected patients.
• Antiretroviral drugs and recommended doses in
hepatic insufficiency.
Management of Patients with Persistently Normal
Aminotransferase
Establishing the persistence of normal aminotransferase
levels in HCV-infected patients is difficult, especially in coinfected patients. Fluctuations in the levels of aspartate
aminotransferase and alanine aminotransferase are common
in this group of patients due to several factors, among
which are the use of drugs of hepatotoxic potential, alcohol
abuse, and infection with other opportunistic agents. In
contrast to mono-infected patients, who present
persistently normal alanine aminotransferase levels (~25%),
co-infected patients present levels of 7-9%. However, of
such patients, 25-40% present advanced liver fibrosis,
which leads to liver cirrhosis.
The rapid evolution of fibrosis in co-infected patients,
even in those with normal transaminase levels, indicates
treatment, based on patient motivation, duration of the disease,
fibrosis stage, and viral load of HCV.
2007;11 (5) Suppl. 1:42-46.
Definition and Quantification of Liver Fibrosis: When and
How?
Various studies have demonstrated the rapidity of liver
fibrosis progression in HIV/HCV co-infected patients. Such
patients, even those presenting little or no fibrosis, should
undergo histological evaluation at least every two years.
Unfortunately, since it is an invasive procedure, liver
biopsy might present complications resulting from technical
performance. Pathologist reports are often made difficult by
the small size of the liver fragments obtained in the biopsy,
which has repercussions for the indication of anti-HCV
therapy.
Despite the disadvantages of liver biopsy, it remains the
principal technique for determining the severity of hepatic
injury.
Noninvasive procedures to assess the level of the liver
fibrosis are currently divided into two categories: imaging
techniques, such as elastometry (FibroScan); and the use of
biochemical markers (Fibrotest, APRISHASTA, FIB-4, and
Forn Index). These procedures are accurate at discriminating
between the absence of fibrosis and advanced fibrosis but
are not very precise at evaluating the intermediate stages of
the fibrosis. They present good predictive value for advanced
liver fibrosis and cirrhosis.
Biochemical marker determination in co-infected patients
is of little utility, given the inflammatory nature of the HIV
disease and the fact that, in this population, hepatotoxic drugs
are used. Such drugs interfere with the serum markers of
fibrosis in various ways: atazanavir elevates bilirubin levels;
non-nucleoside analog reverse transcriptase inhibitors affect
gamma glutamyl-transferase levels; and some protease
inhibitors elevate cholesterol levels.
Due to technical difficulties, complications and the number
of times that the degree of fibrosis must be determined, liver
biopsy might soon be replaced by FibroScan for the evaluation
of co-infected patients, at least in one of their follow-up visits.
Predictors of the Response to Anti-HCV Therapy in CoInfected Patients
The current trend in the treatment of HCV infection is to
individualize the approach.
All HIV/HCV co-infected patients should undergo HCV
genotyping before any therapeutic decision is made.
Since the first attempts at treatment of HCV infection in
HIV-positive patients, a small percentage of patients presenting
a sustained virological response at the end of the treatment
has been demonstrated, especially those with manifested
immunosuppression with low CD4 counts. These patients
present high viral loads of HCV, lower CD8 anti-HCV
responses, greater frequency of liver steatosis caused by the
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Treatment of HIV/HCV Co-Infection
use of alcohol and hepatotoxic drugs (resulting from a lower
kinetic response to HCV treatment), a higher percentage of
adverse effects, and worse treatment compliance.
In order to achieve an early virological response, it is
necessary to interfere with the natural and adaptive responses,
which are reduced in co-infected patients, inducing a delay in
viral clearance, which translates to a late virological response.
High levels of HCV RNA in co-infected patients might also explain
the low rate at which a sustained virological response is achieved.
Approximately 30% of co-infected patients do not present
a significant reduction in HCV viremia during the first month
of treatment with pegylated interferon and ribavirin.
The precise indication for treatment in HIV/HCV coinfected patients is CD4 counts greater than 350 cells/mm3. In
patients with CD4 counts between 200 and 350 cells/mm3, the
decision to treat the HCV infection should take other factors
into account, such as the duration of the HCV infection, the
severity of the liver disease, the degree of HIV suppression,
and the classic predictors of the response to the HCV
treatment (genotype and viral load).
A sustained virological response can be predicted based
on the HCV RNA serum negativity in week 4 of treatment.
However, a reduction of less than 2 log IU/mL in the viral load
of HCV in week 12 or the presence of detectable viremia in
week 24 predicts the absence of a virological response, and
the treatment should be discontinued.
Ongoing studies focus on the maintenance of the treatment
for 72 weeks in co-infected patients who are nonresponsive
at week 24. Perhaps this, or even a longer time with smaller
and fractionated doses, is the real treatment time for HIVpositive patients.
Contraindications and Special Populations
The patients with hepatic decompensation (ascites,
digestive bleeding, hepatic encephalopathy, etc.) cannot be
treated with interferon due to the high risk of developing
serious complications. In these patients, the possibility of a
liver transplant should be considered, even though this
possibility is still remote because of the inherent difficulties
of immunosuppression and HIV infection.
However, the patients with compensated (Child-Pugh class
A or B) cirrhosis can be treated since they are the ones that
benefit most from the treatment.
As to intravenous drug users and chronic alcoholics, the
treatment should be postponed until the habits are controlled,
and such patients should be referred to a detoxification
program.
Therapeutic Doses of Pegylated Interferon and Ribavirin
To date, the efficacy of high doses of pegylated interferon
in the treatment of HIV/HCV co-infected patients has not been
confirmed, and we should therefore await the results of future
investigations.
Ribavirin induces errors in the viral replication cycle, and
this effect is relevant, principally in HIV-positive patients, in whom
43
the mediated immune response is impaired. Various authors have
demonstrated that the dose of ribavirin is directly related to
achieving a sustained virological response. High doses of this
drug are fundamental to the maintenance of viral suppression,
especially in the first weeks of the treatment. Therefore, the
recommended dose for the treatment of HCV is 15 mg/kg/day
(the minimal dose for adjustment is 11 mg/kg/dose).
Treatment Duration
The current consensuses recommend that the duration of
treatment for HIV/HCV co-infected patients be 48 weeks
regardless of the genotype. However, values of HCV RNA > 2
log IU/mL at week 12 have a negative predictive value similar
to that observed for mono-infected patients. Nevertheless,
recent studies have questioned this simplistic view of the
treatment time. When patients infected with genotype 2 or 3
test negative for HCV RNA in week 4 and maintain their
negativity until week 12, the treatment time is reduced to 24
weeks. When patients infected with genotype 1 or 4 are in the
same situation, the duration of treatment is extended to 48 weeks.
For the patients who, despite positivity in week 4, present a
drop in HCV RNA > 2 log IU/mL by week 12, the polymerase
chain reaction (PCR) for qualitative HCV should be repeated in
week 12. If the PCR is negative, and the genotype is 2 or 3, the
treatment should continue to week 48. However, if the genotype
is 1 or 4, the treatment should be maintained until week 72.
If the qualitative PCR for HCV is still positive at week 24,
or there is an HCV RNA reduction < 2 log IU/mL in week 12,
the treatment should be discontinued, regardless of the
genotype.
Using erythropoietin and filgrastim has been a good
strategy, even in co-infected patients, for the treatment of
anemia and neutropenia, respectively.
Treatment of Nonresponsive and Recidivist Patients
At least to date, the HIV/HCV co-infected patients, in
contrast with mono-infected patients, are not candidates for
liver transplant, due to the disease progression and the
absence of a therapeutic response.
A growing number of co-infected patients who have
already undergone treatment with standard interferon with or
without ribavirin, especially those with progression of the
fibrosis staging, can now be retreated with pegylated interferon
and ribavirin.
For the patients who have already used pegylated
interferon and ribavirin and did not respond to the treatment
or presented recurrence, there is currently no regimen for
infection control.
Despite the absence of a virological response in these
patients, 35-43% of them present a reduction in the degree of
liver fibrosis, demonstrating the antifibrotic effect of
interferon. These data provide a rationale if the maintenance
of interferon, even in small doses, for a prolonged time, has
no effect on the progression of the fibrosis, even in the patients
in whom HCV has not been eradicated.
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44
New anti-HCV drugs are urgently needed, especially for this
group of patients, who depend exclusively on clinical treatment.
Treatment of Acute Infection in HIV-Positive Patients
Outbreaks of acute HCV infection in homosexuals have
been reported in some European cities. Despite the knowledge
of the low transmissibility of HCV through sex, sexual practices
that generate traumatic lesions and genital ulcerations have
been associated with the infection.
The natural history of HCV infection in HIV patients has
demonstrated its evolution to chronicity. Therefore, early
therapeutic intervention (in the acute phase of infection) is
particularly indicated in these cases, although treatment should
not be instituted earlier than 12 weeks after the exposure, due
to the possibility of spontaneous viral clearance. However, a
delay in starting treatment might result in a reduction of the
therapeutic response.
The treatment of acute HCV infection in HIV-positive
patients seems to provide a pattern of lower virological
response when compared to HIV-negative patients. However,
the viral clearance pattern obtained in HIV-positive patients
in the acute phase is greater than that seen in those presenting
chronic infection.
Acute infection in HIV-positive patients should be treated
with pegylated interferon and ribavirin for 24 weeks.
HIV/HCV/HBV Co-Infected Patients
In HIV-positive patients living in developed countries, the
prevalence of multiple viral hepatitis (HBV/VCV; VBV/VDV; VBV/
HCV/HDV) is lower than 3%. However, this is still higher than
that seen in the general population. Patients presenting HBV/
HCV co-infection seem to present reciprocal inhibition of viral
replication, with one of the viruses being predominant. However,
this predominance might oscillate from one virus to the other.
Nevertheless, in patients with severe immunosuppression,
replication of all of the viruses might occur simultaneously. In
HIV-positive patients with good immune status, the interference
seems to favor HCV, to the detriment of HBV.
The progression of the liver disease seems to be more
accelerated in HIV-positive patients infected with both HBV
and HCV. In addition, these individuals are more likely to
develop hepatocarcinoma.
Using interferon to treat chronic HDV in HIV-positive
patients is rarely effective.
There is no established consensus as to the approach to
treating multiple viral hepatitis. Few studies have examined
the efficacy and safety of the combination of pegylated
interferon and ribavirin for treating multiple infections in HIVpositive patients. When possible, all of the viruses involved
in the hepatitis should be treated.
Interaction between Antiretroviral Medications and AntiHCV Therapy
The highly active antiretroviral therapy (HAART) regimen
is associated with increased survival due to the fact that it
controls HIV and slows the progression of the hepatic disease.
The better prognosis that the HAART regimen confers on HIV/
HCV co-infected patients is probably due to deceleration of the
progression of hepatic fibrosis. This has principally been
demonstrated in HIV patients treated with protease inhibitors.
Some studies have suggested the early introduction of
antiretroviral therapy in HIV/HCV co-infected patients,
considering the beginning of the HAART regimen in patients
with CD4 counts > 350 cells/mm3, particularly in male patients
and in those who acquired HCV at an advanced age, which
would decrease the effects resulting from immune
reconstitution and the slowing of the progression of HCVinduced hepatic disease.
The principal complication in the treatment of HIV-infected
patients is the interaction between ribavirin and some
antiretroviral drugs.
Ribavirin can decrease the concentrations of some
nucleoside analog reverse transcriptase inhibitors (NRTIs),
although there seem to be no clinical consequences.
The adverse effects of the NRTIs have been associated
with HCV co-infection, female gender, obesity, and prolonged
exposure to these drugs. The principal NRTI combinations
associated with lower impact on the liver are as follows:
lamivudine+abacavir (3TC/ABC); 3TC+zidovudine (AZT);
3TC+ABC+AZT; and emtricitabine + tenofovir. However,
recent studies have indicated that ribavirin interferes with the
effects of ABC, and, therefore, its use in the HAART regimen
for HCV patients should be considered with caution.
The greatest toxicity is that resulting from the concomitant
use of didanosine and ribavirin, worsening with the addition
of stavudine. These combinations increase the possibility of
mitochondrial toxicity and are potentiated by the presence of
HIV and HCV. In patients treated with these antiretroviral drugs
combined with ribavirin, lactic acidosis, pancreatitis and
hepatic decompensation have been reported. Therefore, it is
recommended that replacements for these antiretroviral drugs
be found for patients who will initiate the treatment for HCV.
In addition, AZT should be replaced, when possible, due to
frequent, severe anemia and neutropenia when AZT is
administered together with ribavirin.
It is important to emphasize that the antiretroviral drugs,
as well as the drugs used in the treatment and prophylaxis of
the various opportunistic infections, are metabolized by the
liver and, therefore, many of these metabolites can increase
the risk of hepatic lesion.
The chart below shows the principal interactions between
the drugs used, facilitating the choice of antiretroviral drugs
in the clinical approach to co-infected patients.
The combination of ritonavir and saquinavir has proven
to be highly hepatotoxic, and its use is not currently
recommended, especially in HIV/HCV co-infected patients
with concomitant tuberculosis, in whom noncompliance with
treatment and, principally, the elevation of transaminase levels
result from the discontinuation of the treatment with
antituberculosis drugs or the introduction of alternative
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Chart 1. Drug interactions
Antiretroviral drugs Adverse effects
Therapeutic alternative
Zidovudine
Anemia and neutropenia
Stavudine
Didanosine
Hepatotoxicity
Mitochondrial toxicity
Hepatotoxicity
Mitochondrial toxicity
Stavudine
Nevirapine
Hepatotoxicity
Ribavirin
Hepatotoxicity
Interaction with anti-HCV therapy
Potentiates the probability of anemia
caused by ribavirin
Lamivudine or efavirenz
Ribavirin inhibits the
phosphorylation of this drug
Lamivudine or efavirenz
Should not be combined with
didanosine due to potentiation of
mitochondrial toxicity
Efavirenz
Do not combine with didanosine and
stavudine; potentiation of
mitochondrial toxicity
Tolerated in low doses
Increases the probability of
when used in combination intolerance to the antiretroviral
with other protease inhibitors therapy.
HCV=hepatitis C virus.
Chart 2. Antiretrovirals and recommended doses in liver failure
Name
Hepatic metabolism
Recommendations
Yes
Yes
No
No
Unknown
No
Yes
Mild (A*)=200 mg twice a day / moderate/severe=contraindicated
Not recommended
No need for dose adjustment -recommended
No
Yes
Yes
Yes
Not recommended
Not recommended
Mild (A*)/moderate (B*)=No need for dose adjustment -recommended
Severe (C*)=contraindicated
Protease inhibitors
Atazanavir
Yes
Fosamprenavir
Yes
Indinavir
Nelfinavir
Ritonavir
Yes
Yes
Yes
Mild (A*)=No need for dose adjustment - recommended
Moderate (B*)=300 mg/day. Severe (C*)=contraindicated
Mild/moderate=700 mg 2xday
Severe=contraindicated (because the dose cannot be reduced to
lower than 700 mg)
Mild/moderate=600 mg 3× day. Severe=not recommended
Not recommended
Mild/moderate=No need for dose adjustment -recommended
Severe=not recommended
Mild/ moderate=not recommended. Severe=contraindicated
NRTIs
Abacavir
Didanosine
Emtricitabine
Lamivudine
Stavudine
Zalcitabine
Zidovudine
NtRTIs
Tenofovir
NNRTIs
Delavirdine
Efavirenz
Nevirapine
Saquinavir
Yes
Boosters w/ ritonavir
Atazanavir/ritonavir Yes
Fosamprenavir/ritonavir
Lopinavir/ritonavir Yes
Saquinavir/ritonavir Yes
Tipranavir/ritonavir Yes
Fusion inhibitors
Enfuvirtide
Unknown
Not recommended at some levels of hepatic insufficiency because
it has not been adequately studied
Yes Not recommended
Not recommended
Mild/moderate=Not recommended. Severe=contraindicated
Mild=No need for dose adjustment - recommended
Moderate/severe=contraindicated
Not recommended
*Child-Pugh class. NRTIs=nucleoside analog reverse transcriptase inhibitors; NtRTIs=nucleotide analog reverse transcriptase inhibitors;
NNRTIs=non-nucleoside analog reverse transcriptase inhibitors.
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45
46
regimens that are less efficient in the resolution of infection
with Mycobacterium tuberculosis.
The data currently available are insufficient to determine the
nature of the interaction of atazanavir, tenofovir, fosamprenavir,
and tipranavir with the therapeutic regimen in HCV infection.
References
1. Arends J.E., Boucher C.A.B., Hoepelman A.I.M. Hepatitis C virus
and human imunodeficiency virus coinfected: where do we
stand? Journal of Medicine 2005;63:156-63.
2. Lai, et al. Antiretroviral medication considerations for individuals
coinfected with HIV and hepatitis C virus. AIDS Patient Care
and STDs 2006;20(10):678-92.
3. Rockstroh J.K. Influence of viral hepatitis on HIV infection.
Journal of Hepatology 2006;44:S25-S7.
4. Sancho A.R., Soriano Vicente. Coinfección por el VIH y el virus de
la hepatitis C. http//www.doyma. Acessado em 26/02/2007.
5. Soriano V., et al. Care of patients coinfected with HIV and hepatitis
C virus: 2007 updated recomendations from the HCV-HIV
international panel. AIDS 2007,21:1073-89.
6. Thomas L.D. Options for treatment of hepatitis C in HIV-infected
persons. Journal of Hepatology 2006;44:S40-S3.
www.bjid.com.br
47
Basic Aspects of the Treatment for Hepatitis C: Mechanisms of Action of Interferon Alpha and
Ribavirin and the Bases of Individualization
Carlos Eduardo de Melo, Evaldo Stanislau Affonso de Araújo and Antonio Alci Barone
Laboratory of Hepatitis, LIM 47 DCMIP-HC-FMUSP; São Paulo, SP, Brazil
Pharmacological Characteristics of Interferons and
Ribavirin
The treatment of patients with chronic hepatitis C has
developed considerably in recent years. However, it is still
based on the use of interferon alpha (IFN-α) as an antiviral
and immunomodulatory agent against the hepatitis C virus
(HCV).
The IFNs are a family of proteins that are naturally
produced by the cells of the immune system. The IFN-á protein
presents antiviral, antiproliferative and immunomodulatory
activity [1-3]. Its mechanism of biological action occurs
through the activation of specific genes, influencing cell
growth and division, as well as modulating some immune
system activities. Therefore, IFNs have an indirect antiviral
effect on HCV [2,4].
Commercially, IFN-α is produced by means of recombinant
DNA techniques and is available in preparations of two distinct
subtypes (IFN-α 2a or IFN-α 2b), which can be combined
with other molecules, such as polyethylene glycolor, more
recently, albumin [5,6]. The only difference between IFN-α 2a
and IFN-α 2b is in the amino acid present at position 23 of the
protein: IFN-α 2a has a lysine at that position, whereas IFN-α
2b has an arginine [7].
After the binding with its specific receptor (IFNAR) on
the surface of the target cells, IFN-α activates an intracellular
signaling cascade, which takes the induction of IFN-stimulated
genes (ISGs), establishing a non-virus-specific antiviral state
inside the cell [3,7]. The principal signaling mechanism used
by IFN-α is the so-called Janus kinase/signal transducers and
activators of transcription (Jak/STAT) pathway [3]. Therefore,
two cytoplasmatic proteins with the activity of tyrosine kinase
associated with IFNAR, activated Jak1 and tyrosine kinase 2
(Tyk2), are activated by the dimerization of the receptors.
Activated Jak1 and Tyk2 perform the phosphorylation of
STAT1 and STAT2, respectively. The phosphorylated STAT1
and STAT2 bond with the protein p48 forming IFN-stimulated
gene factor 3 (ISGF3), which translocates into the nucleus
and bonds with IFN-stimulated regulatory element in the
sequences which promote a variety of genes inducible by
IFN-α including antiviral proteins such as 2’5’-oligoadenylate
synthetase (2’5’OAS), protein kinase RNA, and Mx protein
[1,3,7,8].
The absorption of IFN-α (2a or 2b) is high (above 80%)
when administrated intramuscularly or subcutaneously. The
concentration typically peaks at 3-12 h after administration [9].
2007;11 (5) Suppl. 1:47-48.
The metabolism and elimination of IFN-α occurs principally
via the kidneys, with a half-life of 3-8 h [9].
Pharmacological Characteristics of the Pegylated
Interferons
Pegylated IFNs (PEG-IFNs) are produced through the
binding of an inert molecule of polyethylene glycol to the
recombinant IFN-á, thus reducing the renal clearance, altering
the metabolism and increasing the half-life of the IFN molecule,
although maintaining all of its immunostimulatory
characteristics [10,11].
The two PEG-IFNs currently available are produced with
polyethylene glycol molecules of different complexities. PEGIFN-α 2b consists of the binding of IFN-α 2b with a linear PEG
chain, forming a 12-kDa molecule. PEG-IFN-α 2a is formed by
the binding of two 20-kDa chains with IFN-α 2a, resulting in a
complex 40-kDa molecule [6].
The differences in the chemical structure of the two PEGIFN-α formulations are associated with significant
differences in the pharmacological characteristics of the
two drugs. The PEG-IFN-α 2b (12 kDa) is more rapidly
absorbed (with an absorption half-life of 4.6 h), presents a
wide volume of body distribution (approximately 0.99 L/
kg) and a mean elimination time of 40 h. However, PEG-IFNα 2a (40 kDa) is absorbed more slowly (absorption half-life,
50 h), its distribution is restricted to well-vascularized
organs with good perfusion, such as the liver, and it remains
detectable in the serum for one week (approximately 65 h
elimination half-life) [6,12,13].
Pharmacological Characteristics of Ribavirin
Ribavirin is a synthetic nucleoside which is structurally
similar to guanosine [14,15]. Ribavirin enters into the
eukaryotic cells rapidly and, after it undergoes intracellular
phosphorylation, shows virustatic activity against a broad
spectrum of DNA and RNA viruses [14,15].
The exact mechanism of the antiviral action of ribavirin
has not yet been totally elucidated [1,16]. However, some
studies suggest the following possible mechanisms:
a) direct inhibition of HCV replication;
b) inhibition of the enzyme inosine monophosphate
dehydrogenase of the host;
c) induction of mutagenesis in the viral RNA;
d) immunomodulation by the induction of a T helper 1
(Th1)-type immune response Ribavirin is rapidly
absorbed (half-life of approximately 2 h) and widely
distributed throughout the body after its oral
administration; its metabolization occurs principally
via the kidneys [16].
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48
Treatment for Hepatitis C: Basic Aspects
Treatment with IFN-α has as a success-defining
characteristic, progressively more extensive and vigorous
immune stimulation. The more rapid the stimulation is, the
greater are the chances of success. The study of mononuclear
cells ex vivo and in vivo demonstrated that, 3-6 h after the
administration of conventional IFN-α, 516 genes were
upregulated, of which 88 with actions directly linked to immune
functions [17]; the same phenomenon was observed for PEGIFN, also differentiating responders from nonresponders using
the intensity of expression in certain IFN-inducible genes
(2’5’OAS, MX1, IRF-7 and TLR-7), greater in the responders
and lesser in the Afro-Americans [18,19].
The final pathway of the phenomenon triggered in the cell
nucleus is the activation of effector cells. An initial activation
of the innate immunity (natural killer cells) is supposedly
necessary for the early reduction of the viremia – the greater
and the more rapid it is, the more closely it associates with
achieving a sustained virological response (also differentiating
rapid responders from slow responders). Progressively, as of
week 4 of the treatment, the effective immune stimulation induced
by IFN with the respective reduction of the viremia would enable
the specific defense mechanisms (CD4+ and CD8+ cells) which,
in turn, would be in charge of disposing of the residual infected
cells (hepatocytes and extrahepatic cells) [20]. In fact, Pillai et
al. clearly showed that the magnitude and diversity of the cellular
response was associated with early and sustained virological
responses [21], in contrast to other authors who only associated
the Th1-type cellular response with the initial viremia [22]. At
any rate, it is clear that patients presenting a rapid and vigorous
initial response have greater chances of success. However,
patients presenting a slower response need more long-term
stimulation. Therein reside the bases for the individualization
of treatment. It is equally clear that this initial virological
response depends on the gene stimulus induced by IFN-α.
Whether or not these phenomena imply differences associated
with the different types of IFNs used in clinical practice has yet
to be answered. However, initial evidence was provided by the
analysis of the expression of mRNA of inducible IFN genes in
two groups of patients exposed to the two existing types of
PEG-IFN, suggesting that, despite lower plasma exposure, the
patients who used PEG-IFN-α 2b expressed their genes more
vigorously, emphasizing the relevance of the intracellular
environment in the response to HCV treatment [23].
References
1. Feld J.J., Hoofnagle J.H. Mechanism of action of interferon and
ribavirin in treatment of hepatitis C. Nature
2005;436(7053):967-72.
2. Peters M. Actions of cytokines on the immune response and viral
interactions: an overview. Hepatology 1996,23(4):909-16.
3. Wohnsland A., Hofmann W.P., Sarrazin C. Viral determinants of
resistance to treatment in patients with hepatitis C. Clin
Microbiol Rev 2007,20(1):23-38.
4. Souvignet C., Lejeune O., Trepo C. Interferon-based treatment of
chronic hepatitis C. Biochimie 2007;89(6-7):894-8.
5. Chemmanur A.T., Wu G.Y. Drug evaluation: Albuferon-alpha—an
antiviral interferon-alpha/albumin fusion protein. Curr Opin
Investig Drugs 2006;7(8):750-8.
6. Foster G.R. Review article: pegylated interferons: chemical and clinical
differences. Aliment Pharmacol Ther 2004;20(8):825-30.
7. Pestka S. The human interferon alpha species and receptors.
Biopolymers 2000;55(4):254-87.
8. Pawlotsky J.-M. Mechanisms of antiviral treatment efficacy and
failure in chronic hepatitis C. Antiviral Res 2003;59(1):1-11.
9. USP DI® Volume I: Drug Information for the Health Care
Professional [database on CD-ROM]. Version 5.1. Greenwood
Village, Colo: Thomson Micromedex.
10. Reddy K.R., Wright T.L., Pockros P.J., et al. Efficacy and safety
of pegylated (40-kd) interferon alpha-2a compared with
interferon alpha-2a in noncirrhotic patients with chronic
hepatitis C. Hepatology 2001;33(2):433-8.
11. Strader D.B., Wright T., Thomas D.L., et al. Diagnosis,
management, and treatment of hepatitis C. Hepatology
2004,39(4):1147-71.
12. Bailon P., Palleroni A., Schaffer C.A., et al. Rational design of a
potent, long-lasting form of interferon: a 40 kDa branched
polyethylene glycol-conjugated interferon alpha-2a for the
treatment of hepatitis C. Bioconjug Chem 2001;12(2):195-202.
13. Glue P., Fang J.W., Rouzier-Panis R., et al. Pegylated interferonalpha2b: pharmacokinetics, pharmacodynamics, safety, and
preliminary efficacy data. Hepatitis C Intervention Therapy
Group. Clin Pharmacol Ther 2000;68(5):556-67.
14. Graci J.D., Cameron C.E. Mechanisms of action of ribavirin against
distinct viruses. Rev Med Virol 2006;16(1):37-48.
15. Leyssen P., De Clercq E., Neyts J. Perspectives for the treatment
of infections with Flaviviridae. Clin Microbiol Rev
2000;13(1):67-82.
16. Parker W.B. Metabolism and antiviral activity of ribavirin. Virus
Res 2005;107(2):165-71.
17. Ji X., Cheung R., Cooper S., et al. Interferon alfa regulated gene
expression in patients initiating interferon treatment for chronic
hepatitis C. Gastroenterology 2003;37:610-21.
18. Taylor M.W., Tsukahara T., Brodsky L., et al. Changes in gene
expression during pegylated interferon and ribavirin therapy of
chronic hepatitis C virus distinguish responders from non
responders to antiviral therapy. Journal of Virology
2007;81(7):3391-3401.
19. He X.-S., Ji X., Hale M.B., et al. Global transcriptional response to
interferon is a determinant of HCV treatment outcome and is
modified by race. Hepatology 2006;44:352-9.
20. Tang K.H., Herrmann E., Cooksley H., et al. Relationship between
early HCV kinetics and T-cell reactivity in chronic hepatitis C
genotype 1 during peginterferon and ribavirin therapy. Journal
of Hepatology 2005;43:776-82.
21. Pillai V., Lee W.M., Thiele D.L., et al. Clinical responders to
antiviral therapy of chronic HCV infection show elevated
antiviral CD4+ and CD8+ T-cell responses. Journal of Viral
Hepatitis 2007;14:318-29.
22. Aberle J.H., Perstinger G., Weseslindtner L., et al. CD4+ T cell
responses in patients with chronic hepatitis C undergoing
peginterferon/ribavirin therapy correlate with faster, but not
sustained, viral clearance. The Journal of Infectious Diseases
2007;195:1315-9.
23. Silva M., Poo J., Wagner F., et al. A randomized trial to compare
the pharmacokinetic, pharmacodynamic, and antiviral effects
of peginterferon alfa-2b and peginterferon alfa-2a in patients
with chronic hepatitis C (COMPARE). Journal of Hepatology
2006;45:204-13.
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49
Therapeutic Approach to Acute Hepatitis C
Rodrigo Nogueira Angerami 1 and Fernando Lopes Gonçales Júnior 2
Epidemiological Surveillance Unit, Hospital Epidemiology Service, Hospital das Clínicas, State University at Campinas and Campinas
Referral Center for STD/AIDS; 2Study Group on Hepatitis, Infectious Diseases Division, Clinical Medicine Department, School of Medical
Sciences, State University at Campinas; Campinas, Brazil
1
It is estimated that 150 to 200 million individuals are currently
infected with the hepatitis C virus (HCV) [1-3], and that, annually,
there are 3 to 4 million new cases of infection worldwide [4]. In
view of the impossibility of immunoprevention [5] - either through
vaccines or the use of post-exposure immunoglobulin - and the
risk of chronicity in individuals exposed to the disease, which is
estimated to be between 50% and 85% [2,4,6-9,33], the definition
of strategies aimed at early detection and treatment of infected
individuals, preferably during the acute phase of the infection
[2,4], has been widely discussed in recent years.
In individuals presenting symptoms consistent with acute
hepatitis C (AHC), it is known that the possibility of
spontaneous viral clearance, among various factors, depends
especially on a potent initial cellular immune response
mediated by cytotoxic T lymphocytes - initially by HCVspecific CD8+ T lymphocytes and, subsequently, CD4+ T
lymphocytes [10-12]. Other factors associated with a higher
rate of sustained virological response (SVR) are the specific
characteristics of the patients. Individuals who are Asian or
Caucasian, female, young, and HIV-negative, as well as
presenting a rapid drop in HCV RNA levels and presenting
specific human leukocyte antigen class II alleles, together
with the previously mentioned T cytotoxic response, also
respond better to treatment [16,17,33]. Better responses are
also found in cases of infections with shorter incubation
period produced by smaller inoculates. In addition, in a
prospective study conducted by Santantoni et al., a higher
rate of spontaneous viral clearance was observed in patients
infected with genotype 3 [7].
A diagnosis of acute infection with HCV remains a rare
event in clinical practice. In 70-80% of infected patients, AHC
is asymptomatic, and 75% are anicteric [4,8,13-15,33].
Individuals with AHC present elevated serum levels of alanine
aminotransferase (ALT) and aspartate aminotransferase
(AST), with or without a clinical profile of acute hepatitis,
detection of (previously undetectable) HCV RNA and
seroconversion for anti-HCV antibodies. It is estimated that
infection with HCV accounts for only 20% of all diagnosed
cases of acute hepatitis [13]. In the United States, there are
40,000 cases of AHC annually [12], and only a small
percentage of those are clinically diagnosed in this phase.
Considering the high risk of developing chronic hepatitis
C and the favorable evidence, in terms of therapeutic response,
it is fundamental to improve our capacity to properly detect
and treat cases of acute infection with HCV [12].
At-Risk Populations
Currently, it is thought that, in 90% of cases of infection
with HCV, it is possible to identify the associated risk factors
[14].
2007;11 (5) Suppl. 1:49-52.
In the last two decades, especially after serologic triage
was adopted as obligatory for blood donors, and due to the
increased accuracy of serologic tests, there has been a drastic
reduction in the number of new infections with HCV through
blood and blood products. This resulted in significant
changes in the epidemiological pattern of the disease [2,16,18].
However, new infections still occur via parenteral route, and,
less frequently, by sexual or vertical transmission. Currently,
the principal groups at risk for infection with HCV are as
follows: health professionals exposed through cut or
puncture accidents involving sharp objects contaminated
with infectious material; intravenous drug users; individuals
engaging in sex with HCV-positive partners; patients on
hemodialysis; and patients submitted to various invasive
procedures [4,12,14,18-21].
In the United States, intravenous drug users account for
68% of the new cases of HCV infection, compared with 18% for
individuals engaging in sex with HCV-positive partners, 4% for
health professionals exposed through cut or puncture accidents,
1% for individuals infected through other routes, and 9% for
those in whom the source of infection cannot be identified [22].
Currently, it is believed that the risk of vertical transmission is
significantly potentialized in cases of HIV/HCV co-infection, in
which the rate of such transmission can be as high as 17% [37].
Special attention has been given to the occupational risk
of infection with HCV in health professionals, and the
estimated risk of cut or puncture accidents with sharp objects
contaminated with infectious materials ranges from 1% to
2% [5,22].
Although risks, impacts and recommendations are still
controversial, it is estimated that the risk of sexual transmission
ranges from 0% to 3% [34,37], increasing especially in specific
situations, such as co-infection with HIV or other sexually
transmitted diseases, and between homosexual partners.
It is also known that the risk of HCV infection is higher in
individuals with chronic kidney disease under treatment with
hemodialysis. It is believed that, among such individuals
treated in the United States, the prevalence of anti-HCV
reactivity ranges from 15% to 50% [37].
The definition of protocols for clinical and laboratory
periodic follow-up evaluation in these segments at increased
risk is fundamental in order to improve the detection of AHC.
Laboratory Diagnosis
The laboratory techniques most commonly used in the
detection of anti-HCV antibodies are fundamentally based
on third- and fourth-generation enzyme-linked
immunosorbent assays. Such serologic tests have
demonstrated great specificity (> 99%) and sensitivity (95%99%) [38]. However, one of the possible limitations in the
diagnosis of acute infection with HCV results from the
possibility of late seroconversion, occurring at 4 to 10 weeks
after exposure [23]. Regarding detection of anti-HCV
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50
antibodies, we observe 50% to 70% positivity at symptom
onset; in addition, from postinfection month 3 onward,
antibodies are detected in 90% of the cases [38].
However, HCV RNA determination constitutes an
important tool for early detection of acute infections, since
HCV is detectable by 1 to 3 weeks after transmission [23,38].
Immediately after HCV RNA detection and concomitantly with
the appearance of possible signs and symptoms - 2 to 8 weeks
after infection - it is possible to observe increased ALT levels
[38]. Detection of HCV RNA, in the absence of anti-HCV
antibodies, strongly suggests acute infection, especially
when subsequent anti-HCV seroconversion is observed. The
use of transcription-mediated amplification (TMA), together
with other available techniques, constitutes an additional
strategy for early detection of acute HCV infection [23].
Therapeutic Approaches
The high rate of chronicity observed after infection with
HCV justifies the increasing interest in possible therapeutic
approaches in cases of acute infection.
Various approaches have been proposed in literature, all
of which aim at preventing the progression to chronic disease.
Nevertheless, to date, the ideal treatment regimen to be
adopted in clinical practice has not been established
[4,6,8,9,14,18,19,24]. Comparing results from different clinical
trials is difficult for the following reasons: acute hepatitis C is
frequently asymptomatic; different criteria are used in case
definition; samples of patients are small and heterogeneous;
there are no control groups; different types and doses of
interferon (IFN) are used; there are different end points, and
follow-up time varies enormously. Therefore, it is impossible
to define an ideal therapeutic regimen.
When we evaluate the data available in literature, we
observe excellent results in patients treated in the acute phase
of the infection, even under monotherapy [15,16]. The principal
post-treatment outcome measure is the rate at which patients
achieve an SVR, defined as the absence of HCV RNA detected
in serum by qualitative polymerase chain reaction (PCR) at 24
weeks after the end of the treatment [25,26]. Among patients
treated in the acute phase of the infection, this rate ranges
from 37% to 98% [2,4,8,18,20,24], which is higher than the 54%56% observed among patients with chronic hepatitis C, even
among those under combined therapy with pegylated IFN (PEGIFN) and ribavirin (RBV) [25,26].
Various trials evaluate the efficacy of different regimens
using conventional IFN alpha (IFN α) in the treatment of
AHC. The best results to date were those reported by Jaeckel
et al., whose treatment regimen consisted of monotherapy
with IFN α-2b with inducement - 5 million IU/day, s.c., for 4
weeks, followed by 5 million IU, 3 times a week, for 20 weeks
[2]. In that study, the use of IFN, even without RBV, made it
possible to obtain high SVR levels. However, the Jaeckel et
al. study raises the following considerations: 68% of the
patients included in the study were icteric and therefore
presented a higher probability of having self-limited infection,
which could, in part, explain the high SVR rate reported. As
previously mentioned, symptomatic individuals can evolve
to spontaneous viral clearance. In addition, Kamal et al.
observed a higher SVR rate in treated symptomatic individuals
when compared to those asymptomatic individuals treated
with the same therapeutic regimen (96% and 76%,
respectively) [8]. However, Santantonio et al. reported an
SVR rate of 94% among individuals who did not present
spontaneous viral clearance by week 12 after acute symptom
onset, when treated with PEG-IFN α-2b, in monotherapy, for
24 weeks [12]. Such finding corroborates other reports
demonstrating that it is safe to adopt the expected conduct
up to week 21 in order to await possible spontaneous viral
clearance [15,16,20,28,36].
In view of these facts, it should be noted that, among
individuals acutely infected with HCV, those who are
asymptomatic, especially those who are anicteric and present
normal ALT levels, are more likely to present spontaneous
viral clearance [8,12,14,18,20] - typically between weeks 5
and 12 [27,28]. This viral clearance can occur, in rare situations
(in 10%-50% of cases), up to 24 weeks after symptom onset
[8,9,12-15,17,18,21]. Various authors suggest that the treatment
be initiated, at most, 12 to 16 weeks after symptom onset,
avoiding unnecessary exposure to IFN in patients who can
clear HCV spontaneously [4,10,12,18,33].
However, some authors suggest that, in cases of
asymptomatic acute infection, treatment should be
introduced at the time of diagnosis [12].
It should be noted, however, that lower SVR rates have
been observed in cases of late treatment. These rates were
initially described by Nomura et al. [18] who, using IFN α (6
million IU, i.m., daily, for four weeks), reported an SVR in 87%
of early treated individuals - 8 weeks after detection of
symptoms - and in 40% of patients treated after AHC symptom
onset. This study demonstrated that early introduction of
treatment enabled a high SVR rate, even in short-term treatment
regimen. More recently, in a study conducted by Kamal et al.,
the SVR rate in individuals treated after week 20 was 76.6%,
whereas, in individuals who started therapy at weeks 8 and 10,
the rates were, respectively, 95.3% and 93.2% [20].
Similarly to what was observed in the treatment of chronic
hepatitis C [14,29,30], negative HCV RNA at treatment week 1
was associated with a greater probability of achieving an
SVR [31]. In the study carried out by Kamal et al., achieving
a rapid virological response (RVR), defined as HCV RNA
negativity or a ≥ 2 log10 drop in HCV RNA, by treatment week
4, was found to have positive and negative predictive values
of 88% and 98%, respectively, for achieving an SVR. In
another study, involving individuals with AHC treated with
IFN as monotherapy in daily doses for 4 weeks, HCV RNA
negativity in the first week of treatment was found to have a
positive predictive value of 87% for achieving an SVR [18].
The combination of PEG-IFN α and RBV is currently
considered the first-line treatment regimen for patients with
chronic hepatitis C, those infected with genotype 2 or 3 and
treated thusly achieving an SVR at a rate of 82% [20]. However,
the efficacy, safety, duration and appropriate timing of PEG-IFN
treatment in cases of AHC have not yet been well established.
A multicenter, prospective, randomized controlled study,
using PEG-IFN α-2b and involving a 48-week post-treatment
follow-up period, reported SVR rates of 95.3% and 93.2%,
respectively, in groups of patients in which treatment was
initiated at week 8 or week 12 after symptom onset [20]. Studies
that compared treatment results obtained with conventional
IFN to those obtained with PEG-IFN found similarly high
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efficacy in both formulations [4]. Kamal et al., comparing the
efficacy of PEG-IFN α, with and without RBV, reported 85%
and 80% SVR rates, respectively [20]. It should be highlighted,
however, that no evidence justifies the routine use of the
combination with RBV up to the present moment.
In a multicenter study, patients with acute hepatitis C
received PEG-IFN α-2b (in a dose of 1.0-1.5 μg/kg/week)
initiated at the time of diagnosis and continued for 12 weeks;
HCV RNA negativity was found in 87% at treatment week 4,
in 91% at the end of the treatment (week 12), and in 73% at 24
weeks after the end of the treatment [9]. In patients treated
with higher doses of PEG-IFN (≥ 1.2 μg/kg/week for 12 weeks),
the same authors reported an 84% SVR rate, comparable to
that found for 24-week regimens [9]. In a prospective,
uncontrolled study, De Rosa et al., using PEG-IFN α-2b (1.01.6 μg/kg/week, during 12 weeks) initiated immediately after
diagnosis, reported an overall rate of 74%, although higher
rates were reported when higher doses of PEG-IFN were used
(82% when ≥ 1.33 μg/kg/week doses were used) [21].
Similar to what was observed in the treatment for chronic
hepatitis C, compliance to treatment regimen has proven an
important SVR predictive factor in the treatment of AHC. This
was demonstrated in a multicenter study, in which a 71% overall
response rate was found, compared with an 89% rate found in
the population with expected compliance to treatment [32].
When SVR rates were evaluated in difficult-to-treat
populations, two studies involving intravenous drug users
reported SVR rates between 72% and 74% [9.21]. In both
situations, regular follow-up and multidisciplinary approach
were used in order to provide adequate compliance to the
proposed treatment regimen.
Kamal et al. reported that HCV genotype is also an element
to be considered as an SVR predictive factor in patients with
AHC. Therefore, similar to what was reported in cases of chronic
hepatitis C, there were associations among genotypes,
treatment time, and SVR rates, in patients with acute hepatitis
C. Distinct SVR rates were found, according to the genotype
involved: 63.8% for genotype 1; and 100% for genotypes 2
and 3. However, when a prolonged (24-week) treatment regimen
was adopted, an SVR rate of 88% was observed in patients
infected with genotype 1, higher than the 38% and 60% reported
for 8-week and 12-week regimens, respectively [8].
In addition to genotypes other than genotype 1, relevant
positive predictive factors for achieving an SVR include lower
viral load at the start of treatment, early initiation of treatment,
and rapid negative HCV RNA after the initiation of treatment
[8,18]. Calleri et al. reported that, in addition to achieving an
RVR (by week 4 of treatment) and low pre-treatment viremia,
PEG-IFN doses ≥ 1.2 mg/kg/week were positive predictive
factors for achieving an SVR [9].
It is not currently possible to determine the best treatment
regimen for AHC cases. Nor can we establish a minimum
follow-up time to be adopted after the end of the treatment.
Wiegand et al., in a prospective study using biochemical,
ultrasound, and virological monitoring (HCV-PCR, TMA and
RNA detection in peripheral mononuclear cells) of patients
treated with IFN α, in monotherapy, did not report any
evidence of viral recurrence or hepatocytic lesion during the
period studied (mean, 135 weeks; range, 52-224 weeks) [10].
However, as suggested by Alberti et al. [16] in a meta-analysis
51
and subsequently by other authors, the minimum clinical,
biochemical, and virological follow-up time should not be
less than 48 weeks.
Conclusions
High SVR rates have been reported in patients treated for
acute hepatitis C. In view of this, various national and
international consensuses have recommended the treatment
of this infection [6,14,19,34,35]. However, there is no
consensus regarding the proper timing of the initiation of
treatment onset duration of treatment. The best treatment
regimen to be used, as well as the length of the post-treatment
follow-up period, is also debatable.
Taking into account the fact that acute hepatitis C is
generally asymptomatic, serologic triage and often HCV RNA
testing are universally recommended strategies for correct
diagnostic approach of intravenous drug users, patients
infected with HIV, patients on hemodialysis, children of
mothers with HCV, sexual partners of HCV-positive
individuals, and health professionals having been exposed
to HCV. These segments are currently considered the principal
at-risk groups for acute infection with HCV and, therefore,
potentially considered for treatment.
Diagnostic laboratory tests should include anti-HCV
antibody tests (detectable between post-infection weeks 4 and
10), determination of serum levels of aminotransferases such
as ALT (high between post-exposure weeks 2 and 5) and
especially detection of HCV RNA (detectable by 1 week after
infection). The performance of liver biopsy for the diagnosis
of acute hepatitis C is restricted to situations in which the
clinical profile is consistent, although anti-HCV seroconversion
or recent HCV RNA detection are not characteristic.
Individuals with acute hepatitis C are those who present
increased ALT, accompanied or not by clinical profile
consistent with acute hepatitis, with detection of (previously
undetectable) HCV RNA and seroconversion for anti-HCV.
Taking into account the possibility of spontaneous viral
clearance of HCV, especially reported in symptomatic
individuals with acute infection, it is recommended that, in
symptomatic individuals, the treatment be initiated from week
12 after symptom onset onward.
In the case of asymptomatic patients with acute infection,
treatment should be initiated at the time of diagnosis, since
there is less probability of spontaneous viral clearance and
due to the fact that late specific therapy is associated with a
lower SVR rate.
Both IFN formulations - conventional IFN and PEG-IFN are considered efficacious options for the treatment of acute
hepatitis C. However, there has been an increasing tendency
toward using PEG-IFN as a first option, especially in
individuals infected with genotype 1. The option of high
doses of PEG-IFN should be especially considered in
situations in which short-course treatment regimens are used
or in cases of infection with genotype 1. When there is an
option for using the conventional IFN treatment regimen,
adopting a daily induction dose, followed by reduction of
the number of doses, up to the end of the treatment, seems to
be the most efficacious option. There is as yet no conclusive
evidence that the combination of IFN and RBV is superior to
monotherapy with IFN.
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52
Therefore, as observed in studies of chronic hepatitis C
with HCV genotype 1, prolonged treatment regimens are
recommended in cases of acute hepatitis C with HCV genotype
1, although 24-week treatments can also be considered. In acute
infections with genotype 2 or 3, short-course treatments (e.g.,
12-week courses) can be feasible options.
Prolonged (24-week) treatment or combined therapy with
RBV can be considered for patients who do not present an
early virological response in the first 4 weeks of treatment.
Monotherapy regimens, with shorter duration and supervised
administration of IFN, especially in groups of difficult-totreat patients, would be desirable.
The ideal duration of post-treatment evaluation - clinical,
biochemical (ALT levels) and virological (detection of HCV
RNA) - has not yet been well established. However, durations
of at least 48 weeks should be considered.
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13. Afdhal N.H. The natural history of hepatitis C. Seminars in
Liver Diseases 2004;24,suppl. 2:3-8.
14. Strader D.B., Wright T., Thomas D.L., Seeff L.B. AASLD
Practice Guideline – Diagnosis, Management and Treatment
of Hepatitis C. Hepatology 2004;39:1147-71.
15. Heller T., Rehermann B. Acute hepatitis C: a multifaceted
disease. Seminars in Liver Diseases 2005;25:7-17.
16. Alberti A., Boccato S., Vario A., Benvegnù L. Therapy of
acute hepatitis. Hepatology 2002;5:S195-S200.
17. Micallef J.M., Kaldor J.M., Dore G.J. Spontaneous viral clearance
following acute hepatitis C infection: a systematic review of
longitudinal studies. Journal of Viral Hepatitis, 2006;13:34-41.
18. Nomura H., Sou S., Tanimoto H., et al. Short-term interferonalfa therapy for acute hepatitis C: a randomized controlled
trial. Hepatology 2004;39:1213-19.
19. Consensus Statements on the Prevention and Management of
Hepatitis B and Hepatitis C in the Asia-Pacific Region. Journal
of Gastroenterology and Hepatology 2000;15:815-41.
20. Kamal S.M., Fouly A.E., Kamel R.R., et al. Peginterferon alfa-2b
therapy in acute hepatitis C: impact of onset of therapy on
sustained virologic response. Gastroenterology 2006;130:632-8.
21. De Rosa F.G., Bargiacchi O., Audagnotto S., et al. Twelveweek treatment of acute hepatitis C virus with pegylated
interferon-a-2b in injection drug users. Clinical Infectious
Diseases 2007;45:583-8.
22. Alter M.J. Prevention of spread of hepatitis C. Hepatology
2002;36:suppl.1:593-8.
23. Mondelli M.U., Cerino A., Cividini A. Acute hepatitis C:
diagnosis and management. J Hepatol 2005;42:S108-S14.
24. Corey K.E., Ross A.S., Wurcel A., et al. Outcomes and treatment
of acute hepatitis C virus infection in a United States population.
Clinical Gastroenterology and Hepatology 2006;4:1278-82.
25. Manns M.P., Mc Hutchison J.G., Gordon S.C., et al.
Peginterferon alfa-2b plus ribavirin compared with interferon
alfa-2b plus ribavirin for initial treatment of chronic hepatitis
C: a randomized trial. Lancet 2001;358:958-65.
26. Fried M.W., Shiffman M.L., Reddy K.R., et al. Peginterferon
alfa-2a plus ribavirin for chronic hepatitis C virus infection.
N Engl J Med 2002;347:975-82.
27. Hofer H., Watkins-Riedel T., Janata O., et al. Spontaneous
viral clearance in patients with acute hepatitis C can be
predicted by repeated measurements of serum viral load.
28. Gerlach J.T., Diepolder H.M., Zachoval R., et al. Acute
hepatitis C: high rate of both spontaneous and treatmentinduced viral clearance. Gastroenterology 2003;125:80-8.
29. Davis G.I., Wong J.B., McHutchinson J.G., et al. Early virologic
response to treatment with peginterferon alfa-2b plus
ribavirin in patients with chronic hepatitis C. Hepatology
2003;38:645-52.
30. Firenci P. Predicting the therapeutic response in patients with
chronic hepatitis C: the role of viral kinetics studies. J
Antimicrob Chemother 2004;53:15-8.
31. Yamaji K., Hayashi J., Kawakami Y., et al. Hepatitis C viral
RNA status at two weeks of therapy predicts the eventual
response. J Clin Gastroenterol 1998;26:193-9.
32. Wiegand J., Baggish P., Boecher W., et al. Early monotherapy with
pegylated interferon alpha-2b for acute hepatitis infection: the
HEP-NET acute-HCV II study. Hepatology 2006;43:250-6.
33. Jaeckel E., Cornberg M., Waldemeyer H., et al. Acute hepatitis
C: to treat or not to treat? Hepatology 2002;35:1538-40.
34. Management of hepatitis C: 2002. NIH Consensus
Development Conference, 2002.
35. Angerami R.N., Stucchi R., Gonçales N.S.L., Gonçales Jr. F.L.G.
Hepatite C aguda. II Consenso da Sociedade Paulista de
Infectologia para Manuseio e Terapia da Hepatite C 2004:34-6.
36. Zekry A., Patel K., Mc Hutchison J.G. Treatment of acute
hepatitis C infection: more pieces of the puzzle. J Hepatol
2005;42:293-6.
37. Focaccia R., Galante V.C., Oliveira U.B. Hepatite C –
Epidemiologia. In: Tratado de Hepatites Virais. Ed Roberto
Focaccia, 2ª Edição, Editora Atheneu, 2007:211-16.
38. Consenso da Sociedade Paulista de Infectologia para Manuseio
e Terapia da Hepatite C, 2004.
www.bjid.com.br
53
Treatment of Chronic Hepatitis C in Treatment-Naïve Patients
Marcelo Simão Ferreira
Federal University of Uberlândia School of Medicine; Uberlândia, MG, Brazil
After the initial acute phase of infection, 50-85% of patients
infected with the hepatitis C virus (HCV) develop the chronic
form of the disease, which, in 20-30% of cases, will evolve to
cirrhosis, liver failure or hepatocellular carcinoma, albeit after
several decades. Once this infection has been established, it
rarely resolves spontaneously. It is known that, during the
chronic phase, the more severe forms of this viral infection
can be induced by various cofactors: chronic alcoholism; coinfection with HIV or the hepatitis B virus; liver biopsy-proven
steatosis (or steatohepatitis); and advanced age. The main
objective for treating this disease is, therefore, to prevent the
occurrence of late complications, by means of the eradication
of HCV, which can be achieved in just over half of the cases
treated with the currently available drugs.
Treatment for Chronic Hepatitis C: Drugs and Treatment
Response Patterns
The currently recommended treatment for the chronic
forms of hepatitis C is the combination of interferon alpha
(IFN-α) and ribavirin. The former is a cytosine that is a
component of the innate response of the human host. Various
genes involved in the immune response are induced/stimulated
by IFN-α, resulting in the activation of natural killer cells,
maturation of dendritic cells, and proliferation of memory cells,
as well as in the prevention of apoptosis of T cells. The
hepatocellular injury seen in chronic hepatitis C is not due to
the cytopathic effect of HCV. It is immunomediated by natural
killer cells and CD8 T lymphocytes, which are activated by
the action of IFN-α.
Ribavirin is an oral nucleoside analog with antiviral effects
against various pathogens (respiratory syncytial virus,
arenavirus, etc.) Although its mechanism of action in HCV
remains unclear, it seems that ribavirin causes the virus to
mutate rapidly to forms that are more easily killed, as well as
depleting intracellular adenosine triphosphate, which is
essential for the synthesis of viral RNA. Immunomodulatory
effects have been also attributed to this drug.
The use of IFN-α was approved for hepatitis C treatment
in 1991. At that time, the response to this immunotherapy was
very low (< 20%). However, when the combination of ribavirin
and IFN-α began to be used, the proportion of patients in
whom a sustained virological response (SVR) was achieved
grew to 40-45%. Even at that time, treatment responses were
confirmed by detecting HCV RNA through molecular tests
such as polymerase chain reaction (PCR). In this context, the
infection is considered eradicated when there is an SVR, which
is defined as the absence of serum HCV RNA in a sensitive
test (qualitative PCR) at the end of treatment and at six months
2007;11 (5) Suppl. 1:53-57.
after the end of treatment. Patients who achieve an SVR almost
always show a dramatic decrease in HCV RNA levels, defined
as a ≥ 2 log10 drop or the absence of HCV RNA by 12 weeks
after the initiation of treatment. This response is designated
the early virological response (EVR) and has been widely used
for treatment follow-up of patients infected with HCV
genotype 1. The maintenance of the undetectable viral load
status at the end of the treatment is designated the end-oftreatment response (ETR). A patient is considered recidivist
when HCV RNA becomes undetectable during treatment but
becomes positive again after the end of the treatment, whereas
a patient is considered a nonresponder when HCV RNA levels
remain stable or decrease < 2 log10 during treatment with the
combined regimen.
The most recent advance in the treatment of hepatitis C
has been the development of long-acting, pegylated
formulations of IFN-α (PEG-IFN-α), produced by the covalent
addition of a polyethylene glycol molecule to the IFN-α
molecule. This combination decreased absorption, reduced
the clearance of the drug and increased its half-life. With this
increased half-life, PEG-IFN-α can be administered in weekly
doses. Two pegylated formulations have now been approved
for the treatment of hepatitis C: PEG-IFN-α 2a (PegasysRoche), with a molecular weight of 40 kDa, and PEG-IFN-α 2b
(PEG-INTRON; Schering-Plough), with a molecular weight of
12 kDa.
Table 1 shows the names and doses of the drugs that are
currently used for the treatment of chronic hepatitis C.
Viral Kinetics After the Beginning of Treatment
The most important objective of chronic hepatitis C
treatment is HCV eradication. The introduction of treatment
leads to a biphasic drop in the viral population. The speed at
which the quantity of HCV drops differs among the treated
patients, and the drops can therefore be classified as rapid
or slow. Patients in whom there are rapid drops at the
beginning of treatment more often achieve an SVR. During
this rapid response phase, which generally occurs within
the first 48 h of HCV treatment, the viral load decreases
rapidly, which reflects the IFN-α inhibition of replication and
the degradation of the drug in the serum. The HCV RNA
titers begin to decline 8 to 12 h after the administration of the
first IFN-α dose, and the drop ranges from 0.5 to 1.5 log10
within the first 48 h. Thereafter, the rate of viral load reduction
slows, reflecting the clearance of the virus in the infected
cells. The complete elimination of viral particles requires
combined treatment for several months. During this second
phase, ribavirin seems to play a crucial role in HCV
depuration. Negative HCV viral load during treatment is
generally followed by alanine aminotransferase
normalization and improvement in the necroinflammatory
activity in liver biopsy.
www.bjid.com.br
54
Chronic Hepatitis C in Treatment-Naïve Patients
Table1. Drugs used for the treatment of chronic hepatitis C
Drugs
Conventional interferon
Alpha 2a
Alpha 2b
Consensus
Pegylated interferon
Peginterferon α 2a (40 kDa)
Peginterferon α 2b (12 kDa)
Nucleoside analogs
Ribavirin
Recommended doses
3 MU sc 3 × week
3 MU sc 3 × week
9 μg sc 3 × week
180 μg SC / week
1.5 μg/kg SC / week
From 1,000 mg (≤ 75 kg) to
1250 mg (> 75 kg), oral, daily
MU: million units; sc: subcutaneous.
Treatment Protocols
For adults diagnosed with chronic hepatitis C and
presenting detectable serum levels of HCV RNA, together
with persistent elevation of aminotransferases, histological
evidence of progressive hepatic disease, no severe
comorbidities, and no contraindications, treatment is
recommended. All patients should be initially submitted to
viral load quantification (quantitative PCR), HCV genotype
identification (genotypes 1 to 6) and liver biopsy for the
evaluation of necroinflammatory activity (intensity) and
fibrosis (staging). The two most common methods for
histological evaluation are the METAVIR and Ishak scoring
systems, in which fibrosis is scored as absent (F0), only portal
(F1), portal with septum formation (F2), hepatic with portalcentral and portal-portal bridging (F3), or cirrhosis (F4).
Treatment is recommended for patients who present a score
of at least F2.
It must be borne in mind that liver biopsy is an invasive,
costly, and potentially fatal procedure. For patients presenting
HCV genotype 1, this procedure is useful for therapeutic
decisions when there is no evidence of advanced fibrosis
detected by other methods (ultrasound, etc.) However, for
those presenting genotype 2 or 3 and high treatment response
rates, liver biopsy might be unnecessary and might not
influence the therapeutic decision, especially for those
individuals who present persistently high levels of
aminotransferases.
The combination of PEG-IFN-α and ribavirin is the
treatment currently recommended for patients with chronic
hepatitis C; its efficacy in achieving an SVR is greater than
that of the conventional treatment with IFN-α in isolation or
with the combination of IFN-α and ribavirin (56% vs. 16% vs.
42%). As demonstrated in Table 1, subcutaneous PEG-IFN-a
is administered weekly, and oral ribavirin is administered in
two daily doses. The recommended dose of PEG-IFN-α 2a is
180 μg per week and that of PEG-IFN-α 2b is 1.5 μg/kg per
week. Treatment duration and ribavirin dosing vary according
to HCV genotype. Patients presenting HCV genotype 1 must
be treated with either 1,000-mg or 1,250-mg doses of ribavirin
(body weight ≤ 75 kg or > 75 kg, respectively) for 48 weeks.
Patients infected with HCV genotype 2 or 3 must receive 800
mg of ribavirin daily for 24 weeks. In Brazil, ribavirin capsules
contain 250 mg, and so it is not possible to administer the
recommended 800-mg doses. Therefore, we believe it is
prudent to prescribe 1000-mg doses for all patients infected
with these genotypes. There is little information on the
treatment against hepatitis C genotypes 4, 5, and 6 (which are
rare in Brazil). It has been recommended that the 48-week
treatment regimen be used in these cases.
In the treatment for infection with HCV genotype 1, the
possibility of achieving an SVR is based on the EVR, as
previously defined. Negative results or a significant (≥ 2 log10)
drop in HCV viral load by week 12 of treatment is indicative of
an SVR, which appears in 65% of the patients treated with
PEG-IFN-α 2a and in 72% of those treated with PEG-IFN-α 2b.
However, among those who have not achieved an EVR by
week 12 using either formulation, only 3% achieve an SVR.
Therefore, the EVR is a strong negative predictor of the SVR.
All patients who continue to present positivity (based on
viral loads) at week 12 should be re-assessed at week 24, at
which point a qualitative PCR should be performed. If results
are negative, treatment should be maintained up to week 48. If
is the results are still positive, treatment should be
discontinued. For HCV genotypes 2 and 3, it is not generally
recommended that molecular tests be performed during the
24-week treatment regimen. At the end of the combined
treatment, regardless of the genotype, a qualitative PCR should
be performed in order to evaluate the ETR.
Chart 1 shows the algorithm for the follow-up of patients
with chronic hepatitis C during the treatment with PEG-IFN-α
and ribavirin.
With the current treatment protocols, the rate at which an
SVR is achieved ranges from 54% to 56% after the use of the
combination of PEG-IFN-α (either formulation) and ribavirin,
which is considerably higher than that seen with older
conventional treatments (Chat 1). Among patients infected
with HCV genotype 2 or 3, the rate at which an SVR is achieved
ranges from 75% to 80%, compared with 40% to 61% among
those infected with HCV genotype 1. Among patients with
HCV genotype 1, the rate at which an SVR is achieved is lower
in blacks (28%) than in Caucasians (52%). Other factors that
influence treatment response, leading to lower cure rates, are
high viral load levels (> 600,000 IU/mL), male gender, high
body mass index, biopsy showing advanced fibrosis and high
iron levels in the hepatic parenchyma. In patients infected
with HCV genotype 2 or 3 and treated with a conventional
regimen involving the combination of IFN-α (3 million units 3
times a week) and ribavirin, the rate at which an SVR is achieved
can be similar to that of those treated with pegylated
formulations and ribavirin. Therefore, this regimen is still
recommended in Brazil (directive 863/2002). For such patients,
the use of this treatment regimen for six months is less costly
and better tolerated.
Patients with liver cirrhosis have lower chances to eliminate
HCV by means of the current treatment than do noncirrhotic
patients. This might be due to the fact that the former present
more advanced age, greater alterations in hepatic
microcirculation (which prevent the adequate interaction
between IFN and infected cells), and lower treatment
www.bjid.com.br
55
Chart 1. Chronic hepatitis treatment strategies
Table 2. Drugs for the treatment of chronic hepatitis C
Genotype
PEG-IFN-α
α
(dose)
Ribavirin
1
(4, 5, 6)
α 2a: 180 μg/week
α 2b: 1.5 μg/kg/week
1.0 g (< 75 kg)
1.25 g (> 75 kg)
48
Same as above
1.0 g
24
2, 3
Duration Treatment evolution
(dose) (weeks)
a) No response: ¯HCV RNA at week 12
< 2 log10 IU/mL or HCV RNA ⊕ by week 24
b) Rapid response: treat for 24 weeks if
HCV RNA Θ by week 4 and low initial
viral load (< 600,000 IU/mL)
a) No response: uncommon
b) Rapid response: treat for 12-16 weeks
if HCV RNA Θ by week 4
PEG-IFN=pegylated interferon; SVR=sustained virological response; HCV=hepatitis C virus.
www.bjid.com.br
SVR
(%)
40-61
75-80
56
compliance rates. These patients should be treated carefully
due to the risk of decompensation of the disease during
treatment and the worsening of pre-existing hematological
parameters (leukopenia and platelet reduction). Patients with
compensated cirrhosis should be treated if they present the
minimum criteria to receive these medications:
a) Total bilirubin < 1.5 g/dL
b) Albumin > 3.4 g/dL
c) Platelets > 75,000/mm3
d) Hemoglobin > 13 g/dL; neutrophils > 1500/mm3
e) Creatinine < 1.5 mg/dL
f) Absence of ascites, together with hepatic encephalopathy.
In patients with advanced fibrosis and treated with the
PEG-IFN-α/ribavirin combination, the rate at which an SVR is
achieved ranges from 37% to 50%, being higher (70%-75%) in
those presenting HCV genotype 2 or 3, as well as in those
with low viral loads. Side effects, especially thrombocytopenia
and neutropenia, are more common in these patients. At this
stage of the disease, cirrhotic patients who achieved an SVR
are not totally protected against the risk of developing
hepatocellular carcinoma. Two recent studies have confirmed a
limited reduction in the risk of hepatocellular carcinoma in
patients who successfully responded to the combined
regimen; however, some degree of risk remains due to the
carcinogenic effect of hepatic fibrosis. In addition, the
complications of advanced liver disease, in these cases,
occur less frequently, mortality is lower, and there will
obviously be no re-infection of the organ after the liver
transplant. In order to prevent re-infection in transplanted
patients, those who are on a liver transplant waiting list and
present decompensated cirrhosis have been treated with
IFN-α or PEG-IFN-α and ribavirin for periods ranging from
three to fourteen months. In these individuals, the rate at
which an SVR is achieved has been low (from 20% to 25%),
and morbidity/mortality due to the treatment have been
considerably high. Bacterial infections, severe cytopenias,
and even mortality have been reported during treatment.
However, in those patients who responded to treatment, with
the elimination of HCV, there was improvement in liver
function, fewer episodes of decompensation and lower
mortality. Benefits seem to be higher, as expected, for patients
infected with HCV genotype 2 or 3, who traditionally respond
better to the treatment regimen.
Not every patient whose HCV RNA levels become
undetectable during treatment achieves an SVR. In 10% of
the treated patients, this molecular marker reappears in the
serum during treatment, whereas in 20% of the treated patients,
it reappears after the end of treatment (recurrence); in this
context, HCV RNA becomes detectable a few weeks after the
interruption of treatment, and aminotransferase levels again
increase. Recurrence is more common with short treatment or
when there is a delay in achieving negative HCV RNA results.
In those patients who responded to treatment and achieved
an SVR, long-term follow-up evaluations have shown that
HCV RNA results remain negative in more than 95% of these
patients, confirming the cure and the improvement in
histological parameters.
Modifications During the Course of Treatment of Chronic
Hepatitis C
In patients infected with HCV genotype 1, a 72-week
combined regimen (PEG-IFN-α + ribavirin) may be beneficial
for slow responders, who fail to present negative HCV RNA
results by week 4 or week 12 of treatment (EVR). In a randomized
study, 326 patients still presented positive results for the virus
at week 4 of treatment. These patients received the
medications for either 48 or 72 weeks. The rate at which an
SVR was achieved was significantly higher in the group
receiving the longer-duration regimen (45% vs. 32%; p = 0.01).
In another randomized study, patients who did not achieve an
EVR were submitted to either 48 or 72 weeks of treatment. The
results showed that 29% (31/106) of the patients in the 72week group achieved an SVR, compared with only 17% (17/
100) of those in the 48-week group (p = 0.04). Therefore,
selected patients who present slow initial response to antiviral
medications can be treated for longer periods.
However, the treatment duration can be shortened if
patients develop a rapid virological response (RVR), defined
as presenting a response by week 4 of treatment. Various
studies have demonstrated that negative results in the
qualitative PCR by week 4 of treatment have a positive
predictive value for SVR. Studies involving patients infected
with HCV genotype 2 or 3 who achieve an RVR with the use of
PEG-IFN-α 2b and ribavirin have demonstrated that treatment
can be discontinued at 12 to 16 weeks after the beginning of
treatment, since these patients presented SVR rates that were
similar to those seen in the control group, which was submitted
to the 24-week regimen. Recurrences were especially more
common among patients infected with HCV genotype 2 and
in those with high viral loads before treatment. In a recent
randomized study, 150 patients infected with HCV genotype 2
were treated with PEG-IFN-α and ribavirin for either 16 or 24
weeks. The comparison showed that both groups presented
high SVR rates (94% and 95%, respectively), indicating that
patients infected with HCV genotype 2 can be treated for a
shorter period of time. In a similar study, involving patients
infected with HCV genotype 1, good results were
demonstrated using a 24-week course of treatment. Patients
achieving an RVR and a low viral load (< 600,000 IU/mL),
treated for this short period of time, presented a high cure rate
(89%), confirming the hypothesis that, even in patients
infected with the more difficult to treat genotypes, it is possible
that treatment can be shortened if patients are adequately
selected regarding short-term regimens.
Although further studies are necessary to confirm the
efficacy of less prolonged therapies for the treatment of
chronic hepatitis C, these current studies have already
indicated that it is possible to use this strategy for patients
achieving an RVR, significantly improving treatment
compliance and quality of life of the patients, as well as
reducing the frequency of side effects and increasing the
cost-effectiveness ratio. Table 2 summarizes the current
recommendations for the treatment of chronic hepatitis C,
showing the recommended medications and doses, together
with treatment durations and responses.
www.bjid.com.br
Contraindications to and Side Effects of Treatment
Absolute contraindications to the PEG-IFN-α and ribavirin
treatment include pregnancy, breastfeeding and
hypersensitivity to either drug. Relative contraindications,
due to potential side effects are as follows: decompensated
liver disease (jaundice, ascites, hepatic encephalopathy,
severe coagulopathy, etc.); neuropsychiatric, pulmonary,
cerebrovascular or coronary diseases; severe autoimmune
diseases; malignant neoplasms; convulsions; and history
of solid organ transplants. Patients addicted to injection
drugs, as well as chronic alcoholics, should be advised to
abandon the habit (for at least six months) prior to the
beginning of the antiviral therapy. Patients diagnosed with
anemia, leukopenia and platelet reduction should be treated
with care, and hematological parameters should be closely
monitored throughout the treatment.
Side effects of interferon and ribavirin affect practically
all of the patients. Table 3 shows these side effects, which
are organized by their frequency.
The most common side effects are fatigue, myalgia,
psychological alterations (depression, anxiety, insomnia, and
irritability) and worsening of hematological parameters
(anemia, platelet reduction, and leukopenia). Ribavirin
induces hemolytic anemia, which frequently requires dose
reduction. In addition, ribavirin is teratogenic, which requires
strict contraceptive control during treatment. Only 1% to
2% of patients will develop severe side effects, requiring the
interruption of one or both medications. Recombinant
erythropoietin (for anemia) and filgrastim (for neutropenia)
should be routinely used for the control of treatment-induced
cytopenias. Patients suffering from depression or mood
disorders can use antidepressants or anxiolytics, with
variable success.
References
1. Hoofnagle J.H., Seef L.B. Peginterferon and Ribavirin for chronic
hepatitis C. New England Journal of Medicine 2006;355:244451.
2. Strader D.B., Wright T., Thomas D.L., Seef L.B. Diagnosis,
management and treatment of hepatitis C. Hepatology
2004;4:1147-71.
3. Iacobellis A., Siciliano M., Perri F., et al. Peginterferon alfa-2b
and ribavirin in patients with hepatitis C virus and
decompensated cirrhosis: a controlled study. Journal of
Hepatology 2007;46:206-12.
4. Bruno S., Stroffolini T., Colombo M., et al. Sustained virological
response to Interferon a is associated with improved outcome in
HCV-related cirrhosis: a retrospective study 2007;45:579-87.
5. Schiff E.R. Emerging strategies for pegylated interferon
combination therapy. Gastroenterology & Hepatology
2007;4(1):517-21.
6. Navasa M., Forns X. Antiviral therapy in HCV decompensated
cirrhosis: to treat or not to treat? Journal of Hepatology
2007;46:185-8.
7. Everson G.T., Hoefs J.C., Seef L.B., et al. Impact of disease severity
on outcome of antiviral therapy for chronic hepatitis C: lessons
from the HALT-C trial. Hepatology 2006;44:1675-84.
8. Tan J., Lok A.S.F. Update on viral hepatitis: 2006. Current Opinion
in Gastroenterology 2007;263-7.
57
Table 3. Side effects of interferon and ribavirin
Side effects
Interferon α (including PEG-IFN)
Ribavirin
Flu symptoms (greater with PEG-IFN-α 2a)
Neutropenia
Thrombocytopenia
Depression, acute psychosis,
suicide attempts
Irritability
Visual disorders
Fatigue, myalgia
Hypothyroidism/hyperthyroidism
Headache
Nausea, vomiting
Itching
Fever, weight loss
Hearing loss
Alopecia
Pulmonary interstitial fibrosis
Angina/myocardial infarction
Bacterial infections (in cirrhotic patients)
Hemolytic anemia
Fatigue
Pruritus
Skin rash
Sinusitis
Fetal malformations
Gout
PEG-IFN=pegylated interferon.
9. Hadzyiannis S.J., Sette H., Morgan T.R., et al. Peginterferon a 2a
and Ribavirin combination therapy in chronic hepatitis C. Annals
of Internal Medicine 2004;140:346-55.
10. Pawlotsky J.-M. Current and future concepts in hepatitis C
therapy. Seminars in Liver Disease 2005;25:72-83.
11. Mangia A., Santoro R., Minerva N., et al. Peginterferon alfa 2b
and Ribavirin for 12 vs 24 weeks in HCV genotype 2 or 3. The
New England Journal of Medicine 2005;325:2609-17.
12. Zenzem S., Buti M., Ferenci P., et al. Efficacy of 24 weeks treatment
with chronic hepatitis C infected with genotype 1 and low
pretreatment viremia. Journal of Hepatology 2006;44:97-103.
13. Delgard O., Bjoro K., Hellum K.B., et al. Treatment with pegylated
interferon and ribavirin in HCV infection with genotype 2 or 3
for 14 weeks: a pilot stydy Hepatology 2004;40:1260-5.
14. Manns M., Waldemeyer H., Cornberg M. Treating viral hepatitis C:
efficacy, side effects and complications. Gut 2006;55:1350-9.
15. Davis G.L. Monitoring of viral levels during therapy of hepatitis
C. Hepatology 2002;36:5145-51.
16. Manns M.P., McHutchison J.G., Gordon S.C., et al. Peginterferon
alfa 2b plus ribavirin for initial treatment of chronic hepatitis
C: a randomized trial. Lancet 2001;358:958-65.
17. Hofmann W.P., Zeuzem S., Sarrazin C. Towards individualized
antiviral therapy of patients infected with hepatitis C virus
genotypes 2 and 3. Hepatology Reviews 2006;3:3-10.
18. Abergel A., Hezode C., Leroy V., et al. Peginterferon alpha-2b
plus ribavirin for treatment of chronic hepatitis C with severe
fibrosis: a multicentre randomized controlled trial comparing
two doses of peginterferon alpha 2b. Journal of Viral Hepatitis
2006;13:811-20.
19. Hung C.H., Lee C.M., Lu S.N., et al. Long term effect of interferon
alpha 2b plus ribavirin therapy on incidence of hepatocellular
carcinoma in patients with hepatitis C virus-related cirrhosis.
Journal of Viral Hepatitis 2006;13:409-14.
20. EASL International Consensus Conference on hepatitis C. Paris,
26-27 February 1999. Consensus statement. Journal of
www.bjid.com.br
58
Retreatment of Hepatitis C Patients Who Previously Experienced Treatment Failure
Fernando Lopes Gonçales Jr.
Department of Viral Hepatitis Studies, State University of Campinas (Unicamp); Campinas, SP, Brazil
In its different formulations, interferon (IFN) alpha
combined with ribavirin (RBV) is the best treatment alternative
for patients infected with the hepatitis C virus (HCV) [1]. In
such patients, the objective is to achieve an end-of-treatment
virological response – negative serum HCV ribonucleic acid
(RNA) – followed by a sustained virological response (SVR),
which is defined as HCV RNA negativity for six months after
the suspension of treatment.
The treatment regimens for HCV infection have evolved
from monotherapy with conventional IFN alpha to combined
therapy with IFN and RBV and, more recently, to combined
therapy with pegylated IFN (PEG-IFN alpha-2a or alpha-2b)
and RBV [2,3]. It is known that approximately 60% of the
patients infected with HCV genotype 1 and nearly 40% of
those infected with genotype 3 do not achieve an SVR when
treated with the conventional regimen of IFN and RBV [2-7].
The availability of PEG-IFNs has reduced the percentage of
patients experiencing treatment failure. In large international
trials involving treatment-naïve patients, regimens that include
the combination of PEG-IFN and RBV have produced
significantly higher rates of SVR than those observed with
the use of the conventional combination of IFN and RBV (5456% vs. 44-47%) [5-7].
Similar to what occurs in treatment-naïve patients,
retreatment provides the patient with a new chance to achieve
an SVR. Long-term studies have proven that the vast majority
of the patients who achieve an SVR usually remain negative
for HCV RNA for a long time [8].
The arguments in favor of retreating patients would be
the possibility of eradicating HCV, reducing fibrosis, and
decreasing the risk of evolving to hepatocarcinoma. With
regard to the new antiviral agents, we do not know when they
will be available or whether all patients will be able to wait
several years to initiate retreatment.
It is known that patients who experience relapse after
treatment with conventional IFN, with or without RBV, respond
better to retreatment with PEG-IFN alpha and RBV than do
those presenting no response to treatment with conventional
IFN (with or without RBV). Krawitt et al. observed an SVR rate
of 55% in 66 relapsing patients who were retreated with PEGIFN alpha-2b (100-150 µg/week) and RBV (1000 mg/day),
compared with only 20% in 116 previous nonresponders
treated with the same regimen [9]. An SVR was observed in
53% of the relapsing patients infected with genotype 1,
compared with 59% of the relapsing patients infected with
genotypes 2 or 3. There were, therefore, no significant
differences between these groups of patients. This was not
2007;11 (5) Suppl. 1:58-59.
observed in the previous nonresponders receiving retreatment.
Of those, only 17% of the individuals infected with genotype
1, in comparison with 57% of the individuals infected with
genotypes 2 or 3, achieved an SVR. Therefore, genotype had
an influence on SVR in the previous nonresponders who
underwent retreatment.
Studies conducted in Brazil and involving nonresponders
to IFN/RBV demonstrated SVR rates resulting from retreatment
with the PEG-IFN alpha-2a + RBV regimen [10], as well as with
the PEG-IFN alpha-2b + RBV regimen [11], that were higher
than those reported in international studies. Of the relapsers
treated, 57% [10] and 62% [11] achieved an SVR. These SVR
rates in relapsers are higher than, for example, the rates of
41% and 59% obtained, respectively, in Canada (by Sherman
et al.) and in France (by Moucari et al.) [12,13]. In analyzing
the response in relapsers by genotype, the Brazilian
researchers found that an SVR was achieved in 69-70% of
those infected with genotype 3, compared with 43-44% of
those infected with genotype 1 [10,11].
True nonresponders, that is, patients who never presented
viral negativity during or at the end of the treatment, are the
most difficult group to retreat. Some authors showed that,
after previous treatment with conventional IFN, with or without
RBV, relapsers have significantly higher SVR when retreated
with PEG-IFN alpha-2b and RBV than do nonresponders to
previous treatment (55% vs. 20%; p<0.001) [9]. The same was
observed by Sherman et al., who found that, after retreatment
with PEG-IFN alpha-2a and RBV, the SVR rate was 23% in
nonresponders and 41% in relapsers [12]. The results of
the retreatment of true nonresponders to PEG-IFN and RBV
obtained by the Brazilian researchers are better than those
reported in international studies [10,11]. However, it is very
difficult to compare different studies with different
populations of patients treated with different formulations
of conventional IFN.
Two large international trials (EPIC3 and HALT-C) are
evaluating, in two different international cohorts, the
responses to treatment with the combination of PEG-IFN and
RBV in nonresponders to previous treatment with IFN and
RBV [14,15]. The EPIC3 study is also evaluating the real
meaning of the virological response at week 12 and its
predictive value in nonresponders. The final results of both
trials will soon be available.
With regard to the use of induction doses of PEG-IFN in
the first 12 weeks or the increase in the duration of the
treatment of nonresponders, there are few studies in the
literature.
In a recent pilot study, researchers working in Spain
observed that patients with genotype 1 HCV who were
retreated for 72 weeks, with different induction doses of PEG-
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Retreatment of Hepatitis C Patients
IFN alpha-2a in the first 12 weeks, achieved an SVR at a rate
of 30-37%, which is practically twice as high as the SVR rate
of 18% observed in those treated with normal doses of PEGIFN alpha-2a [16].
Since nonresponders to initial treatment/relapsers
constitute a very heterogeneous group, it is necessary to
carefully qualify and select the patients who should be
retreated. The various factors that might have been
responsible for the lack of a response should be modified/
nullified prior to the initiation of or even during the new
treatment cycle. Currently, the patients who are considered
the least likely to respond to retreatment are the true
nonresponders, as well as those who are black, infected
with genotype 1, presenting a high viral load, having
advanced liver disease, or presenting intercurrent diseases,
such as obesity, HIV coinfection, and coinfection with
hepatitis B virus.
Those who were previously treated with IFN as
monotherapy or with the combination of IFN and RBV are
more likely to achieve an SVR than are the nonresponders
to the combination of PEG-INF and RBV. Those who had
relapses during (breakthrough) or after the previous
treatment also fare better than do those who are true
nonresponders. Patients who were noncompliant with the
previous treatment or those who needed to undergo IFN or
RBV dose reduction due to cytopenias or other adverse
effects usually respond better to retreatment than do those
who received full doses. It is clear that the factors
responsible for noncompliance should be corrected. In
addition, medication dose reductions should be adequately
addressed. In nonresponders, erythropoietin and filgrastim
should be used earlier and further dose reductions should
be avoided at all costs. Drug and alcohol users who, due to
their dependence, did not satisfactorily complete all phases
of the previous treatment might respond better to
retreatment if these cofactors are removed. This also applies
to the patients who did not have adequate social or cultural
support. Obese patients, insulin-resistant patients,
dyslipidemic patients, patients with steatosis, and patients
with liver diseases, such as hemochromatosis, should be
retreated, preferably after the diagnosis and treatment of
these accompanying conditions have been appropriately
addressed. The doses of PEG-IFN should be the same as
those used in treatment-naïve patients. We recommend that,
in retreatment, the doses of RBV be as high as possible.
Due to the paucity of studies of large population samples,
the duration of retreatment should be 48 weeks for all
genotypes. At the moment, there are no conclusive data in
the literature to support the use of higher doses of PEGIFN and RBV, the use of induction doses, or the extension
of treatment duration to more than 48 weeks in such
patients. With regard to the week-12 rule, there is strong
evidence that patients who do not present HCV RNA
negativity by that time are much less likely to achieve an SVR,
and that their treatment should therefore be discontinued.
59
References
1. National Institutes of Health Consensus Development Conference
Statement: Management of hepatitis C: 2002-June 10-12,
2002. Hepatology 2002;36:S3-20.
2. McHutchison J.G., Gordon S.C., Schiff E.R., et al. Interferon
alfa-2b alone or in combination with ribavirin as initial
treatment for chronic hepatitis C. N Engl J Med
1998;339:1485-92.
3. Poynard T., Marcellin P., Lee S.S., et al. Randomised trial of
interferon alpha-2b plus ribavirin for 48 weeks or for 24
weeks versus interferon alpha-2b plus placebo for 48 weeks
for treatment of chronic infection with hepatitis C virus.
Lancet 1998;352:1426-32.
4. Poynard T., McHutchison J., Goodman Z., et al. Is an “a la
carte” combination interferon alfa-2b plus ribavirin regimen
possible for the first line treatment in patients with chronic
hepatitis C? Hepatology 2000;31:211-8.
5. Fried M.W., Shiffman M.L., Reddy K.R., et al. Peginterferon
alfa-2a plus ribavirin for chronic hepatitis C virus infection.
N Engl J Med. 2002;347:975-82.
6. Manns M.P., McHutchison J.G., Gordon S.C., et al.
Peginterferon alfa-2b plus ribavirin compared with interferon
alfa-2b plus ribavirin for initial treatment of chronic hepatitis
C: a randomised trial. Lancet 2001;358:958-65.
7. Hadziyannis S.J., Sette H., Morgan T.R., et al. Peginterferonalpha2a and ribavirin combination therapy in chronic
hepatitis C: a randomized study of treatment duration and
ribavirin dose. Ann Intern Med 2004;140:346-55.
8. M a r c e l l i n P. , B o y e r N . , G e r v a i s A . , e t a l . L o n g - t e r m
histologic improvement and loss of detectable intrahepatic
HCVRNA in patients with chronic hepatitis C and sustained
r e s p o n s e t o i n t e r f e r o n - a l p h a t h e r a p y. A n n I n t M e d
1 9 9 7;127:875-81.
9. Krawitt E.L., Ashikaga T., Gordon S.R., et al. Peginterferon
alpha-2b and ribavirin for treatment-refractory chronic
hepatitis C. J Hepatol 2005;43:243-9.
10. Parise E., Cheinquer H., Crespo D., et al. Peginterferon alfa-2
a (40 KD) (PEGASYS) plus ribavirin (COPEGUS) in
retreatment of chronic hepatitis C patients, nonresponders
and relapsers to previous conventional interferon plus
ribavirin therapy. Braz J Infect Dis 2006;10:11-6.
11. Gonçales F.L. Jr., Vigani A., Gonçales N., et al. Weight–based
combination therapy with peginterferon alpha-2b and
ribavirin for naïve, relapser and non-responder patients with
chronic hepatitis C. Braz J Infect Dis 2006;10:311-6.
12. Sherman M., Yoshida E.M., Deschenes M., et al. Peginterferon
alfa-2a (40KD) plus ribavirin in chronic hepatitis C
p a t i e n t s w h o f a i l e d p r e v i o u s i n t e r f e r o n t h e r a p y. G u t
2006 ;55:1631-8.
13. Moucari R., Ripault M.P., Oules V., et al. High predictive value
of early viral kinectics in retreatment with peginterferon and
ribavirin of chronic hepatitis C patients non-responders to
standard combination therapy. J Hepatol 2007;46:596-604.
1 4 . Shiffman M.L., Di Bisceglie A.M., Lindsay K.L., et al.
Peginterferon alfa-2a and ribavirin in patients with chronic
hepatitis C who have failed prior therapy. Gastroenterology
2004;126:1015-23.
1 5 . Poynard T., Schiff E., Terg R., et al. Sustained virologic
response (SVR) in the EPIC3 trial: week 12 virology predicts
SVR in previous interferon/ribavirin treatment failures
receiving Peg-Intron/Rebetol weight based dosing. J Hepatol
2005;42 (Suppl. 2):40.
1 6 . Diago M. Peginterferon alfa-2a (40kd) (Pegasys®) and
ribavirin (Copegus®) in patients infected with HCV
genotype 1 who failed to respond to interferon and ribavirin:
final results of the Spanish high-dose induction pilot trial.
Presented at the 55 th AASLD; October 29-November 2,
2004; Boston, MA.
www.bjid.com.br
60
Maintenance Treatment for the Modulation of Liver Fibrosis
In many cases, the evolution of chronic infection with the
hepatitis C virus (HCV) is favorable. However, progression to
liver fibrosis is a common phenomenon and can lead to liver
cirrhosis. Phenomena associated with liver fibrosis have been
previously reviewed and are more related to the host than to
viral factors [1]. In brief, the onset of fibrosis is caused by the
activation of stellate cells, which acquire the shape of
myofibroblasts and become the source of collagen deposition,
as well as of protein matrix formation. Activated stellate cells
rapidly undergo apoptosis, after which the collagen matrix is
degraded and removed by the activity of metalloproteinases.
There is a complex balance between the procollagen and
antifibrotic factors, although the mechanisms of fibrosis
regression are not fully understood [2]. However, it appears
that the tissue inhibitor of metalloproteinase-1 [3] and stellate
cell apoptosis [4] are crucial to maintaining this balance. The
mechanism through which HCV triggers fibrosis is little
understood. Apparently, hepatocyte infection triggers a state
of oxidative stress and induces inflammatory cell recruitment.
These phenomena lead to the activation of stellate cells and
collagen deposition. In addition, HCV proteins directly activate
stellate cells [4].
Liver cirrhosis and the preceding transition stage are
marked phenomena in the clinical evolution of patients and
have been associated with morbidity/mortality due to chronic
hepatitis C. A study involving a cohort of patients chronically
infected with HCV, monitored from 1991 onward, with a mean
infection period of 22 years, provided evidence that, five years
after the diagnosis of cirrhosis (Ishak score ≥ 4), the survival
rate was 80% [5]. This rate decreased to 19% after the first
hepatic decompensation [5]. In the multivariate analysis,
treatment with the combination of conventional or pegylated
interferon alpha with ribavirin was found to be favorably
associated with survival. In other words, the treatment was
associated with the most important outcome: survival! In
addition, subjects presenting a virological response, whether
sustained or not, presented better evolution when compared
to nonresponders. This fact suggests that even transitory
negative viremia levels are a favorable phenomenon and may
imply lower structural alterations and activity in the liver [5].
One relevant aspect of this study was that the interruption of
alcohol consumption from the moment of diagnosis on
prevented the previous ingestion from having an unfavorable
influence on the disease progression. This fact reinforces the
importance of alcohol abstinence for patients with hepatitis
C. Poynard et al. [6] retrospectively analyzed data from 3,010
patients treated with either conventional or pegylated
2007;11 (5) Suppl. 1:60-63.
interferon alpha, using various therapeutic regimens . The
authors concluded that the treatment with pegylated interferon
and ribavirin resulted in significant improvement in histology,
inflammatory activity and structural alterations (fibrosis).
There was improvement in the ‘fibrosis progression rate’,
which is a valid concept, although reproducibility was
jeopardized by the risks of sample variation. The least
worsening of fibrosis was found in the optimized group
receiving pegylated interferon and ribavirin (8%), and greatest
degree of such worsening was found in the group receiving
interferon for 24 weeks (23%) [6]. In general, fibrosis stabilized
or improved even in those patients not achieving a sustained
virological response (SVR). However, sample variations, a high
percentage of patients with mild initial fibrosis (over 70%
classified as F1), lack of paired biopsy results from all
participants, lack of a control group, and principally, a relatively
short follow-up period (20 months between biopsies, on
average) were limitations of this analysis. Nevertheless,
Poynard et al. [6] addressed the concept of cirrhosis
‘reversion’, or as the authors designated it, the ‘reversible
cirrhosis stage’. This group was composed of young patients,
whose structural staging changed, regressing from F4. This
phenomenon occurred in 75 (49%) of the 153 cirrhotic patients
[6]. The authors postulated that this stage of fibrosis would
still be ‘easily’ reversed. Some of the factors that were found
to be associated with the regression of fibrosis after treatment
are, obviously, the initial degree of fibrosis, minimal baseline
activity, achieving an SVR, being less than 40 years of age,
initial viral load lower than 3.5 million copies/mL, and (a new
concept at that time) body mass index (BMI) < 27 kg/m2 [6]. In
another relevant meta-analysis, Cammà et al [7,8]. evaluated
three randomized clinical studies comprising 1441 patients,
paired biopsies being available for 1013 (70.3%). Similarly to
Poynard et al. [6], these authors demonstrated improvement
in hepatic fibrosis in the patients treated with pegylated
interferon alpha-2a, when compared to those treated with
conventional interferon, who achieved an SVR or even
experienced recurrence. However, there was no improvement
among nonresponders. The authors also found that a BMI
higher than 30 kg/m2 was associated with the worsening of
hepatic fibrosis. However, in a more representative sample –
447 (44%) of 1013 cirrhotic patients – no ‘regression of
cirrhosis’ was found. Only 33% of the cirrhotic patients
presented improvement in fibrosis. However, the observation
period between biopsies was also short. Finally, high alanine
aminotransferase level was another factor that was associated
with histological improvement. More recently, Di Marco et al.
[9] prospectively evaluated cirrhotic patients with portal
hypertension and no previous decompensation who received
pegylated interferon alpha-2b (1.0 μg/kg/week) with or without
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Maintenance Treatment for Liver Fibrosis
ribavirin (0.8 g/day). Patients infected with genotype 2 or 3
showed rapid response, with negative viral RNA results by
week 4 of treatment, and achieved an SVR. However, only 10
of the patients infected with genotype 1 or 4 achieved an
SVR. Nevertheless, for this subgroup of patients, the
predictability of early response by week 12, but not by week
4, was also valid, and high baseline viral load was a negative
predictive marker for SVR. The occurrence of cytopenias was
high; however, stimulating factors were not used with these
patients. If stimulating factors had been available, the SVR
rate associated with treatment maintenance would have
certainly been better. Treatment compliance is even more
critical for this group of patients and was associated with
SVR [9]. Finally, it was clear that SVR is associated with better
evolution. Only 6% of the patients achieving an SVR
deteriorated, compared with 38% of the nonresponders [9].
Bruno et al. [10] conducted a retrospective multicenter study
comprising 920 patients with compensated cirrhosis who were
treated with conventional interferon alpha (from 3 to 6 million
IU/3 times a week) for a year. Similarly to the prospective
study conducted by Di Marco et al. [9], the authors
demonstrated benefits for those who achieved an SVR, with a
reduction in the risk of decompensation, occurrence of
hepatocellular carcinoma (HCC) and death. They also found
that a platelet count of 109,000/mL is an independent predictor
of decompensation of the liver disease [10]. This factor is
extremely relevant for clinical practice and could be used as a
cut-off point in order to define the use of more aggressive
measures, or even those that still need to be validated.
Specifically regarding HCC, Cammà et al. [7,8] in 2001, pointed
out that the benefit of the reduction in the incidence of HCC,
albeit modest, is also more relevant among those who achieve
an SVR after being treated with interferon alpha. Other authors
have also reproduced data regarding the effective and
tolerable treatment [11], fewer complications, reduction or
negative incidence of HCC among cirrhotic patients monoinfected with HCV who achieved an SVR after the treatment
with interferon alpha [12-14], and even histological response
of patients co-infected with HIV [12]. Finally, even patients
with severe cirrhosis under individualized, ascending-dose
regimens benefit from treatment and can achieve an SVR, albeit
a modest one [15-17]. Despite the innumerable benefits of the
treatment with interferon alpha, we must emphasize that
surveillance regarding the incidence of HCC is highly
recommended, even in cirrhotic patients who have achieved
an SVR [10].
There is clear evidence of improvement in fibrosis (a
quantitative decrease, as well as functional gains such as
lower portal hypertension) [3], greater survival, potential
decrease in the incidence of HCC and hepatic complications,
and even ‘cirrhosis reversion’ [6], even for nonresponders [6]
or for those who presented recurrence [5-8]. Therefore, we
should ask why we do not treat even those patients with more
severe cirrhosis [15-17], and why we do not use the treatment
with interferon alpha for objectives other than the virological.
61
In order to answer these questions, it is initially important
that recent knowledge on viral kinetics and the concepts of
treatment individualization – dose and duration –be
considered in the therapeutic decision-making, and that the
same concepts are not clearly validated for cirrhotic patients.
Therefore, if our goal is to achieve an SVR, extending the
treatment of nonresponders to at least 24 weeks of ‘ideal’
treatment is considered ‘futile’ [1]. However, is this true from
a histological point of view, or from the perspective of the
need to modulate the natural history of the disease? In order
to answer these questions and in view of the previously
described evidence, physicians began to consider the use of
maintenance treatment with interferon alpha. However, for a
conclusive analysis, a prospective evaluation would be ideal,
since differences in methodologies would greatly affect the
quality of the results [17]. Three principal studies (Table 1)
have addressed this issue: the Hepatitis C Antiviral LongTerm Treatment against Cirrhosis (HALT-C) trial, conducted
by the NIH; the Evaluation of Peg-Intron in Control of
Hepatitis C Cirrhosis (EPIC)3 trial; and the Colchicine versus
Peg-Interferon Long-Term (COPILOT) trial. Other, smaller,
studies, such as the PROFIC-C trial, have also addressed this
issue [18].
The COPILOT study evaluates patients with fibrosis
classified as greater than Ishak 3 and previous nonresponders
to interferon/ribavirin or pegylated interferon/ribavirin,
comparing, in two branches, colchicine to pegylated interferon
alpha-2b at 0.5 μg/kg/week. Preliminary analyses after a twoyear follow-up period revealed that the group using interferon
presented significantly fewer hepatic complications,
especially portal hypertension and upper gastrointestinal tract
bleeding [6]. Alterations in HCV quantification were minimal.
The EPIC3 trial has yet to produce preliminary results.
However, the HALT-C study has provided a consistent amount
of information. Nevertheless, data regarding the main objective
of the study, fibrosis modulation, are still unavailable but
should be presented at the upcoming congress of the American
Association for the Study of Liver Diseases (AASLD; Afdhal,
personal communication). Using the available results from
HALT-C, Everson et al. [15], in 2006, emphasized the need to
‘optimize’ the treatment of cirrhotic patients, for whom the
SVR rate was lower, regardless of platelet counts or the need
to reduce interferon doses – or even the influence of previous
treatment response. Therefore, cirrhosis is a determining factor
of a lower SVR rate. Among the therapy ‘optimization’ measures
that are currently available, should we consider treatment prior
to the establishment of cirrhosis? What would be the criteria?
We still cannot answer that, but if we consider some preliminary
results of new therapies, we can predict that interferon alpha
will still be the backbone of hepatitis treatment for many years.
Therefore, we should certainly attempt to gain a better
understanding of the potential of these treatments and use
them wisely.
Among the minor studies, Erhardt et al. preliminarily
showed that the maintenance of a 0.35-1.0 μg/kg/week dose
www.bjid.com.br
62
Table 1. Maintenance studies with pegylated interferon
Study
HALT-C
COPILOT
EPIC3
Disease stage
Ishak 4-6
CTP ≤ 6
1400
Placebo, IFN-α, Peg-IFN-α-2a (90 μg)
Peg-IFN á-2b (0.5 μg)
3.5 years
Ishak 3-6
CTP ≤ 7
800
Peg-IFN-α-2b (0.5 μg)
METAVIR 2-4
CTP ≤ 6
1700 (700 cirrhotic)
4 years
3-5 years
Patients (n)
Treatment arms
Colchicine (0.6 mg bid)
Duration
HALT-C=Hepatitis C Antiviral Long-Term Treatment against Cirrhosis (trial); COPILOT= Colchicine versus Peg-Interferon Long-Term
(trial); EPIC3=Evaluation of Peg-Intron in Control of Hepatitis C Cirrhosis (trial) 3; CTP=Child-Turcotte-Pugh score; IFN=interferon;
Peg=pegylated.
of pegylated interferon alpha-2b significantly reduced, after
48 weeks, the incidence of HCC and complications due to
cirrhosis when compared to the control group. In a similar
study, Kaiser et al. [19] demonstrated that, in the intervention
group, the fibrosis score dropped from 3.58 to 2.59 after 18
months of treatment, and to 2.36 by six months after the end
of treatment. In the control group, the fibrosis score increased
from 3.88 to 4.07 and to 4.79 by the same time points. Therefore,
monotherapy with lower doses of pegylated interferon alpha2b effectively reduced and modulated hepatic fibrosis. The
mechanism for the improvement induced by interferon is
unknown and is certainly multifactorial. The elimination of
the triggering agent is undoubtedly crucial, but, since even
patients who suffer recurrence get better, it is possible that
interferon alpha has an intrinsic antifibrotic effect, as well as
inhibiting the activation of stellate cells [4]. Other possible
approaches that are positively associated with the
improvement of hepatic fibrosis in nonresponders – or as a
coadjuvant – are the use of renin-angiotensin inhibitors
(inhibiting the activation of stellate cells) and the control of
metabolic syndrome [4]. Other substances that are potentially
active against hepatic fibrosis are interleukin-10 and the natural
herb known as Sho-saiko-to [4]. The maintenance treatment
with ribavirin is clearly a discarded alternative since there are
no positive effects [20]. Finally, the use of controlled
phlebotomies may be an alternative in selected cases.
Excessive iron in the hepatocytes, increased by the effect of
HCV, has been associated with greater tissue damage [20].
The field of antifibrogenesis is in full development and has
been recently reviewed in an AASLD symposium [3].
Considering all the reviewed aspects, we should finally
evaluate who would benefit from the maintenance treatment.
These would be the patients with extensive fibrosis (F3/F4) or
cirrhosis that does not respond to the optimal standard
treatment. In view of the risk of disease progression and the
onset of HCC, as well as the body of evidence available, simply
monitoring the progression of the disease is not an acceptable
approach, neither for the patient nor for the physician [20].
Therefore, what is the ideal dose and how long should we
maintain the treatment with interferon? The period has yet to
be defined, and it may be indefinite (or until the appearance of
a definitely efficacious, safe therapy). Biopsy monitoring every
two years in association with HCC and screening for
esophageal varices are acceptable standards, although
normalization of alanine aminotransferase levels and HCV
reduction will rarely be seen [20]. The advent of noninvasive
methods of monitoring fibrosis [21,22] has transformed
maintenance into an even more alternative strategy. Regarding
the adopted dose, in ongoing and published studies,
pegylated interferon alpha has been used in smaller-thanhabitual doses, once a week. Tolerance and safety have proven
adequate. Doses from one-third to one-half of the standard
size seem to be satisfactory for hepatic fibrosis modulation.
However, definitive results, which are still unavailable, could
alter this perception.
In summary:
1. Hepatic fibrosis is a potentially reversible
phenomenon, which can even provide functional
benefits.
2. Even for cirrhotic patients, treatment with interferon
alpha is possible, efficient and safe, although it is less
efficacious if we consider the SVR.
3. Treatment with pegylated interferon alpha, even when
it is ineffective, is associated with greater survival,
fewer complications and lower incidence of HCC.
4. Interferon alpha has a modulatory effect on hepatic
fibrosis.
5. Maintenance treatment with reduced doses of
pegylated interferon alpha proved to be effective in
modulating hepatic fibrosis as well as in altering the
natural history of the disease.
6. Antifibrogenesis is a field that is still in development,
and, in addition to interferon alpha, other measures
can be adopted, such as control of metabolic syndrome
and the use of renin-angiotensin inhibitors.
Considering the reviewed aspects, together with the facts
that the number of antiviral therapies currently in development
is smaller than expected, and that none of those are yet
clinically available, as well as the fact that they will still need
to be combined with interferon alpha, I believe that the
optimization of interferon alpha will continue to be crucial. A
pharmaco-economic analysis must be certainly considered as
a supporting tool in the collective decision-making. However,
considering the physician-patient relationship, some
reflections are pertinent and should be debated by the
Hepatitis Committee of the Brazilian Society of Infectology.
www.bjid.com.br
Therefore, we propose the following:
Individualization
The dose and duration of treatment with the combination
of pegylated interferon and ribavirin should be defined based
on patient body weight and on the early response during
therapy for patients with minimal lesions (F1) who nevertheless
are at risk for progression: moderate peri-portal activity, from
40 to 60 years of age and comorbidities (nonalcoholic
steatohepatitis, HIV infection or metabolic syndrome).
Maintenance Treatment
Considering maintenance treatment with pegylated
interferon alpha for patients with structural lesions greater
than F3 or portal hypertension signs (esophageal varices,
enlarged spleen, dilation of vessels) or platelet counts lower
than 110,000/mm3, classified as Child-Pugh class A or B, no
evidence of severe or potentially uncontrollable
decompensation, with no HCC, no recurrence, partial
responders or nonresponders to pegylated interferon alpha
in association with ribavirin (or only to interferon when there
are contraindications regarding ribavirin) whose compliance
can be confirmed for at least 12 weeks. This treatment should
also be considered for patients who present contraindications
regarding the use of full doses. Child-Pugh class C patients
could be treated in specialized centers when on a transplant
waiting list.
Proposed regimen*: subcutaneous pegylated
interferon alpha-2b at 0.5-1.0 μg/kg/week;
subcutaneous pegylated interferon alpha-2a at 90 μg/
week.
Duration*: at least 24 months, indefinite, or until the
appearance of a definitely efficacious, safe antiviral
therapy.
Follow-up evaluation: Monitoring the onset of
complications (HCC, digestive tract hemorrhage,
encephalopathy, etc.)
*Dose schedule and duration were suggested based on preliminary data
and should be reassessed depending on the results of ongoing studies.
References
1. Hoofnagle J.H., Seef L.B. Peginterferon and ribavirin for chronic
hepatitis C. New England Journal of Medicine
2006;355(23):2444-51.
2. Arthur M.J.P.Reversibility of liver fibrosis and cirrhosis following
treatment for hepatitis C.Gastroenterology 2002;122(5):15258.
3. Friedman S.L., Rockey D.C., Bissel M. Hepatic fibrosis 2006:
report of the third AASLD single topic conference. Hepatology
2007;45:242-9.
63
4. Bataller R., Brenner D.A. Liver fibrosis. The Journal of Clinical
Investigation 2005;115:209-18.
5. Lawson A., Hagan S., Rye K., et al. The natural history of hepatitis
C with severe hepatic fibrosis. Journal of Hepatology
2007;47:37-45.
6. Poynard T., McHutchinson J., Manns M., et al. Impact of pegylated
interferon alfa-2b and ribavirin on liver fibrosis in patients with
chronic hepatitis C. Gastroenterology 2002;122:1303-13.
7. Cammà C., Di Bona D., Schepis F., et al. Effect of peginterferon
alfa-2a on liver histology in chronic hepatitis C: a meta-analysis
of individual patient data. Hepatology 2004;39:333-42.
8. Cammà C., Giunta M., Andreone P., Craxì A. Interferon and
prevention of hepatocellular carcinoma in viral cirrhosis: an
evidence-based approach. Journal of Hepatology 2001;34:593602.
9. Di Marco V., Almasio P.L., Ferraro D., et al. Peg-Interferon alone
or combined with ribavirin in HCV cirrhosis with portal
hypertension: a randomized controlled trial. Journal of
Hepatology 2007 (in press).
10. Bruno S., Stroffolini T., Colombo M., et al. Sustained Virological
Response to interferon-á is associated with improved outcome
in HCV-related cirrhosis: a retrospective study. Hepatology
2007;45:579-87.
11. Helbling B., Jochum W., Stamenic I., et al. HCV-Related advanced
fibrosis/cirrhosis: randomization controlled trial of pegylated
interferon á-2a and ribavirin. Journal of Viral Hepatitis
2006;13:762-9.
12. Sarmento-Castro R., Horta A., Vasconcelos O., et al. Impacto f
peginterferon alpha-2b and ribavirin treatment on liver tissue
in patients with HCV or HCV-HIV co-infection. Journal of
Infection 2007;54:609-16.
13. Veldt B.J., Saracco G., Boyer N., et al. Long term clinical outcome
of chronic hepatitis C patients with sustained virological
response to interferon monotherapy. Gut 2004;53:1504-8.
14. Coverdale S.A., Khan M.H., Byth K., et al. Effects of interferon
treatment response on liver complications of chronic hepatitis
C: 9-year follow-up study. American Journal of
Gastroenterology 2004.
15. Everson T.G., Hoefs J.C., Seef L.B., et al. Impact of disease severity
on outcome of antiviral therapy for chronic hepatitis C: lessons
from the HALT-C Trial. Hepatology 2006;44:1675-84.
16. Everson T.G., Trotter J., Forman L., et al. Treatment of advanced
hepatitis C with low accelerating dosage regimen of antiviral
therapy. Hepatology 2005;42:255-62.
17. Everson G.T. Maintenance Interferon for chronic hepatitis C:
more issues than answers? Hepatology 2000;32:436-8.
18. Schuppan D., Krebs A., Bauer M., Hahn E.G. Hepatitis C and liver
fibrosis. Cell Death and Differentiation 2003;10:S59-S67.
19. Kaiser S., Hass H., Luize B., et al. Long term, low dose treatment
with pegylated interferon alfa 2b leads to a significant reduction
in fibrosis and inflammatory score in chronic hepatitis C
nonresponder patients with fibrosis or cirrhosis.41st EASL, 2006.
20. Kelleher T.B., Afdhal N.H. Maintenance therapy for chronic
hepatitis C. Current Gastroenterology Reports 2005;7:50-3.
21. Afdhal N.H., Kowdley K.V., Llovet J.M. CCO Independent
Conference Coverage of the 2007 Annual Meeting of EASL.
Interim analisys: peginterferon alfa-2b maintenance therapy
may reduce incidence of HCV-related HCC, Cirrhosis
complications, 2007.
22. Afdhal N.H., Nunes D. Evaluation of liver fibrosis: a concise review.
American Journal of Gastroenterology 2004;1160-70.
www.bjid.com.br
64
Treatment of Patients Infected with Hepatitis C Virus and Presenting Extrahepatic Manifestations
Fátima Mitiko Tengan1 and Antonio Alci Barone
University of São Paulo School of Medicine; São Paulo, SP, Brazil
HCV and Mixed Cryoglobulinemia
Antiviral treatment should be performed with the same
medications (standard or pegylated interferon alpha (PEGIFN-α, with or without ribavirin) and similar regimens, until
additional controlled studies provide further information on
the treatment of mixed cryoglobulinemia (MC) related to
infection with the hepatitis C virus (HCV).
Data regarding antiviral treatment of MC (Table 1) show
that this therapeutic approach should be the first choice due
to the antiproliferative and immunomodulatory effects of IFN
and the usefulness of antiviral treatment, as demonstrated in
most studies. In addition, the strict correlation between
virological and clinical response, as well as the positive effect
of inhibiting viral replication in the expanded B-cell clones,
which is considered the pathogenic basis of MC, are reasons
to make this choice. However, IFN-α can also trigger or worsen
autoimmune diseases [6,7].
Renal insufficiency and neuropathies can occur or be
worsened, and ulcer cicatrization may be prolonged. Therefore,
treatment with IFN-α should be restricted to symptomatic
patients, with or without renal involvement, after the careful
evaluation of clinical and laboratory characteristics regarding
autoimmunity during this period.
In comparison with the antiviral treatment of chronic
hepatitis C, the antiviral treatment of MC is more complex due
to various reasons, such as the lack of standardized treatment
protocols, the higher incidence of recurrence and the
contraindications to antiviral treatment (old age, severe hepatic
disease, acute nephritis and disseminated vasculitis). In
addition, the interpretation of laboratory findings seems to be
more complex than in chronic HCV infection. In fact,
biochemical markers of MC response (cryocrit, rheumatoid
factor or complement activity) can be more independent of
the virological response than are alanine aminotransferase
(ALT) levels.
At the moment, antiviral treatment is suggested as the
treatment of choice for this condition, even when there is no
indication of hepatic pathology. Patients with apparently
benign manifestations of the disease (palpable purpura,
arthralgia and mild fatigue) should not be treated or can be
symptomatically treated with nonsteroidal anti-inflammatory
drugs. Special attention must be given to the treatment of
patients with severe MC (with acute nephritis and
disseminated vasculitis).For these cases, the data are
insufficient to guarantee the safety of IFN administration, and
we therefore strongly suggest a cautious approach. It is
preferable to use an alternative therapeutic approach to all
patients for whom antiviral treatment is contraindicated or
not tolerated, as well as for those who did not respond to
previous treatment. Possible alternatives include the use of
corticosteroids, immunosuppressants, plasmapheresis and a
hypo-antigenic, or low-antigen-content, diet [8].
Treatment should be individualized for each patient
according to the severity of clinical symptoms, considering
other factors involved (age, comorbidities, etc.) and for a
limited period of time (weeks or months) until the remission of
symptoms. Any therapeutic approach must be avoided for
clinically asymptomatic patients.
Table 1. Therapeutic regimens used for mixed cryoglobulinemia associated with hepatitis C virus infection
Treatment
(months)
Treatment duration
response
End-of-treatment
Sustained
virological response
3 M IU IFN 3×/week
+ RBV
27 NR or 3 M IU IFN 3×/week
Relapsers + RBV
18
3 M IU IFN 3×/week
or PEG-IFN + RBV
9
1.5 μg/kg/week PEG-IFN
+ RBV
18
1.0 μg/kg/week PEG-IFN
+ RBV
6
78%
NA
12
85%
NA
≥ 18
NA
70%
Author
Year
Number
Zuckerman [1]
2000
9 NR
Mazzaro [2]
2003
Alric [3]
2004
Cacoub [4]
2005
Mazzaro [5]
2005
≥ 10
12
88%
89%
44%
CS: corticosteroid; NR: Nonresponders; IFN: interferon; PEG: pegylated; RBV: Ribavirin; NA: not available.
2007;11 (5) Suppl. 1:64-68. © 2007 by The Brazilian Journal of Infectious Diseases and Contexto
www.bjid.com.br
Treating Extrahepatic Manifestations of Hepatitis C
Prior to the identification of HCV, corticosteroid therapy
was the treatment of choice for MC, since corticosteroids,
even in small doses, control most of the symptoms. However,
corticosteroids can favor HCV replication, cause various side
effects and not induce significant changes in the cryocrit or
in the natural history of the disease. Cytostatic,
immunosuppressive drugs (e.g., cyclophosphamide,
chlorambucil and azathioprine) are mainly used, in combination
with plasmapheresis, when there is no response to
corticosteroids and during acute phases of MC (acute
nephritis evolving to renal insufficiency and hyperviscosity
syndrome). Various studies showed that rituximab (anti-CD20
antibody, a specific B-cell surface antigen) is efficacious for
most patients with MC, with the significant improvement or
resolution of MC – particularly skin lesions – and regression
of clonal expansion of B cells [9].
Plasmapheresis is indicated for the removal of circulating
cryoglobulins and immunocomplexes.
Due to its efficacy and fast action, plasmapheresis is
especially recommended in the presence of acute
manifestations (cryoglobulinemic nephritis, severe sensorymotor neuropathies, skin ulcers and hyperviscosity syndrome).
Combined with cyclophosphamide, it has been shown to
effectively reduce the rebound effect at the end of apheresis.
The low-antigen-content diet has low macromolecule content
with high antigenic properties, resulting in more efficient
removal of cryoglobulins by the reticuloendothelial system.
This diet can improve the minor manifestations of the disease
(purpura, arthralgia and paresthesias) and is generally
prescribed during the initial phase of the disease.
HCV and Lymphoma
The inclusion of antiviral treatment seems to be rational in
therapeutic regimens for non-Hodgkin’s lymphomas (NHLs)
and HCV infection. This approach is supported by recent
studies on low-grade lymphomas [10], and, in particular, on
marginal zone lymphomas [11,12].
Vallisa et al. [10] treated 13 patients diagnosed with
concomitant low-grade NHL-B and HCV infection,
characterized by an indolent evolution, with PEG-IFN and
ribavirin. A hematologic response was seen in most patients
(complete and partial response, 75%), and this was strongly
associated with the clearance or reduction of HCV viral load
in serum, after the treatment, which proved to be useful for
treating this pathology.
Hermine et al. [11] reported that patients with concomitant
HCV infection and splenic lymphoma with villous lymphocytes
presented complete remission after being treated with IFN.
The inclusion of a control group with patients diagnosed with
the same disease but presenting no HCV infection
demonstrated that, unlike the patients with HCV infection,
the HCV-negative patients did not respond to the treatment
with IFN. Similarly, remission of polyclonal proliferation in
response to the antiviral treatment proved to be clearly
associated with virological response [13].
65
Although antiviral response seems to be an attractive tool
for low-grade NHL and positive HCV, chemotherapy might be
necessary for intermediate- or high-grade NHL, and the
antiviral treatment can be maintained after chemotherapy [14].
The use of rituximab in NHL associated with HCV, alone or in
combination (with antiviral treatment or with chemotherapy),
seems very promising, especially for low-grade NHL [15-17].
Despite the limited number of described cases, it is reasonable
to consider rituximab a safe and efficacious treatment for
indolent B-cell lymphomas accompanied by HCV infection.
HCV and the Presence of Autoantibodies
From a clinical point of view, the major concern is
represented by the use of IFN. It has been reported that the
administration of IFN can have a negative effect on
autoimmune hepatitis. Significant increases in ALT activity –
even if transitory, corrected with corticosteroids and not
associated with deterioration of liver function – have also
been reported in HCV/anti-liver-kidney-microsomal type 1
(anti-LKM1)-positive cases treated with IFN [18,19].
Initial treatment for CS is recommended when there are
high antibody titers (≥ 1:320), high globulin titers, anti-LKM1
antibodies, anti-human microsomal cytochrome P450 (CYP)
IID6 257-279 antibodies and interface hepatitis with various
plasmocytes. In the case of initial treatment with IFN, rigorous
monitoring of ALT levels is suggested, especially in patients
who are anti-LKM1-positive.
HCV and Sjogren’s Syndrome
In one study, 12 patients diagnosed with concomitant
Sjögren’s syndrome and HVC infection were treated with IFN
alone or with the combination of IFN and ribavirin [20]. Half of
the patients presented improvement of the dry syndrome using
the associated regimen, but none responded to IFN in
isolation. Various patients presented adverse immunologic
events during treatment.
HCV and Arthritis
Treatment decisions must be made case by case. Etiologic
treatment with IFN-a and ribavirin is recommended when there
is hepatic or systemic involvement, since it can occasionally
induce or worsen autoimmune disturbances. The treatment
leads to a significant improvement in HCV-related arthritis,
even without a complete biochemical or virological response.
Cryoglobulinemia-related arthritis generally responds to
antiviral treatment. Considering there are few data available at
the moment, the usually non-aggressive evolution of HCVrelated arthritis does not justify the use of antiviral medications
as a standard treatment.
HCV and Porphyria Cutanea Tarda
Treatment with IFN-a seems to be less efficacious in
patients with concomitant chronic HCV infection and
porphyria cutanea tarda than in those with chronic HCV
infection alone. [21]. The disease also responds to iron
www.bjid.com.br
66
depletion by phlebotomy, which can be performed prior to
antiviral treatment. However, porphyria can be triggered in
genetically predisposed patients treated with the association
of IFN-a and ribavirin, as a consequence of hemolysis
induced by ribavirin, which causes an increase in serum iron
levels [22].
HCV and Lichen Planus
Doutre et al. [23] reported improvement in lesions of two
patients treated with IFN-α. Other authors [24-28] also
reported that lesions disappeared during treatment with IFNá for several months after the end of treatment. Protzer et al.
[29] reported oral and cutaneous lichen planus (LP)
exacerbations during the treatment with IFN-α. Treatment was
discontinued because local measures did not improve the
lesions.
It is generally recommended that stricter control measures
be taken when patients with previous manifestations of LP
receive IFN. There are no detailed reports on the effect of the
combination of IFN and ribavirin in patients with concomitant
LP and HCV infection.
HCV and Thyroid
The treatment with IFN-α may trigger the formation of
autoantibodies in patients with HCV and exacerbate thyroid
dysfunction in patients with pre-existing antibodies [30-33].
Changes are generally detected after three months of treatment
and disappear when treatment is discontinued [34].
In severe cases, treatment must be discontinued,
particularly in patients with hypothyroidism. Alternatively, in
patients previously receiving thyroid medications, it can be
useful to increase the dose during the antiviral treatment [35].
Low antibody titers are not an indication for treatment
discontinuation. The possibility of good treatment control
generally allows the continuity of antiviral treatment. Prior to
treatment, levels of thyroid hormones, including thyroidstimulating hormone (TSH), as well as anti-thrombopoietin
antibodies, must be monitored. In addition, it is opportune to
perform regular TSH monitoring during treatment. When there
are altered values, the decision of continuing or discontinuing
the treatment must be made case by case.
HCV and Type 2 Diabetes Mellitus
Considering that hyperglycemia was considered an
independent risk factor for the ‘nonresponse’ to antiviral
treatment of chronic hepatitis C [36], and that abnormal glucose
levels can be associated with host-related factors, such as
age, gender, alcohol use, ethnicity, obesity and resistance to
insulin, it is recommended that glucose levels in blood be
controlled (by means of medications or changes in lifestyle),
prior to the administration of antiviral treatment.
However, during the acute phase of kidney disease (when
renal insufficiency and systemic manifestations are present),
it is recommended that antiviral treatment be avoided or
discontinued. In this case, measures aimed at reducing the
inflammatory activity of renal lesions (corticosteroid therapy),
removing circulating cryoglobulins (plasmapheresis) and
reducing the formation of new antibodies (cyclophosphamide
administration) are used [41-45]. Under these conditions,
favorable outcomes have been achieved with mycophenolate
mofetil and, more recently, with anti-CD20 antibody (rituximab).
Regarding the use of ribavirin, lower proteinuria and improved
kidney function have been reported in patients treated with
IFN + ribavirin [1, 46,47]. It must be highlighted that ribavirin
clearance is reduced in patients with renal insufficiency, and
that dialysis does not eliminate the medication. Therefore, the
use of this antiviral drug in standard doses is not
recommended for patients who present with creatinine
clearance < 50 mL/min.
HCV and Neuropathies
Treatment with IFN-a is not efficacious and can worsen
peripheral neuropathy in patients with chronic hepatitis C
and MC [48]. A detailed review of available studies on patients
with both HCV infection and peripheral neuropathy was
inconclusive. There were patients who responded to
corticosteroids, endovenous immunoglobulin or
plasmapheresis combined with antiviral treatment [49].
HCV and Resistance to Insulin
When treating patients with HCV infection, the physician
has the challenge of differentiating patients with good
prognoses from those with poor prognoses, especially
regarding the intervention measures available. At the moment,
the available data allow us to infer that the treatment of insulin
resistance (reducing hyperinsulinemia) and of those factors
that surely contribute to the onset and maintenance of
steatosis can increase the rate at which a sustained virological
response is achieved in patients with HCV infection who were
treated with the combination of PEG-IFN and ribavirin.
Comments
Decisions regarding the treatment of the abovementioned
pathologies must be taken on a case-by-case basis, since the
pathogeneses of most of these clinical manifestations are
unknown. Cumulative knowledge on each of the pathologies
must be considered regarding the immunomodulatory effect
of IFN-α, which is the standard treatment for HCV infection.
One option would be the use of an immunosuppressive agent
in conjunction with interferon when there are autoimmune
phenomena.
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47. Reed M.J., Alexander G.J., Thiru S., Smith K.G. Hepatitis Cassociated glomerulonephritis—a novel therapeutic approach.
Nephrol Dial Transplant 2001;16:869-71.
48. Scelsa S.N., Herskovitz S., Reichler B. Treatment of
mononeuropathy multiplex in hepatitis C virus and
cryoglobulinemia. Muscle Nerve 1998;21(11):1526-9.
49. Ramos-Casals M., Trejo O., Garcia-Carrasco M., Font J.
Therapeutic management of extrahepatic manifestations in
patients with chronic hepatitis C virus infection. Rheumatology
[Oxford) 2003;42(7):818-28.
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69
Hepatitis C Treatment Before and After Liver Transplant
1
Edson Abdala 1,2 , Daniela Rosa Magalhães Gotardo1 , Patrícia Rodrigues Bonazzi1,2 and Telésforo Bacchella 1
Liver Transplant Section, Department of Gastroenterology, University of São Paulo School of Medicine; 2Department of Infectious and
Parasitic Diseases, University of São Paulo School of Medicine; São Paulo, SP, Brazil
Advanced hepatic disease, either in the form of cirrhosis
or hepatocellular carcinoma, caused by infection with the
hepatitis C virus (HCV), is currently the main indication for
liver transplant worldwide [1,2]. Hepatitis C also appears as
an etiologic factor for terminal hepatic disease. However,
although this procedure is defined as a standard therapy in
both situations, recurrence of HCV infection is universally
recognized. The potential for HCV infection to evolve in a
more aggressive manner is greater among transplant patients
than among immunocompetent individuals, and the reestablishment of hepatic cirrhosis in these patients can occur
within five to ten years after the transplant. The progression
to cirrhosis also occurs at a more accelerated pace in these
patients, with decompensation rates higher than 40% a year
after diagnosis [3].
Despite the risk of recurrence, cirrhosis caused by HCV
has long represented a disease with good post-transplant
evolution potential and low recurrence rates. In the mid 1990s,
there was an increase in the rate of recurrence, which impairs
the function of the graft and reduces the survival of the patient.
Studies to determine the risk factors began to be carried out,
and the adoption of certain measures has enabled better
outcomes [4].
Natural History of Hepatitis C After Transplant
Early recurrence of the HCV infection after the transplant,
defined as the detection of HCV RNA in the serum or graft, is
a practically universal event, observed in more than 95% of
the cases. Hepatic disease recurrence is represented by a wide
range of histopathological aspects, and the differential
diagnosis with acute cellular rejection can delay its detection.
In these cases, there is overlapping of histopathological
standards, as well as immunopathogenic phenomena in
common [5,6].
In the recurrence context, acute hepatitis generally occurs
between one and six months after the liver transplant, at a
frequency of approximately 70%. Its histopathological
findings are characterized by hepatocyte edema, large-droplet
steatosis, moderate lobular inflammation, and acidophilic
corpuscles. Although spontaneous resolution of acute
hepatitis C occurs in up to 15% of immunocompetent
individuals, it is rarely observed in the transplant context [3,4].
Severe progressive cholestatic hepatitis can occur early,
between one and three months after the transplant. This kind
of recurrence is rarer, occurring only in 10% of the cases. Its
severe evolution pattern is characterized by high levels of
2007;11 (5) Suppl. 1:69-73.
serum bilirubin (over 6 mg/dL), high serum levels of HCV RNA,
central ballooning in the liver biopsy, low inflammatory
infiltrate, and cholangiolar proliferation, without associated
ductopenia, suggesting that HCV has a direct cytopathic effect.
These patients evolve to rapid graft loss, and death occurs
even before a new transplant attempt can be made [4].
In most cases, however, the hepatitis C recurrence is
diagnosed as chronic hepatitis, with a more accelerated
progression of fibrosis than that observed in the
immunocompetent population, resulting in cirrhosis in 8% to
30% of patients within five years. Cirrhosis is also more
aggressive in these patients, with a 65% cumulative risk of
complications within three years. The histopathological
findings found in the graft are similar to those found in the
native liver of an individual with hepatitis C and include mixed
portal infiltrate with lymphoid aggregates, periportal
inflammation, varied lobular inflammation, and steatosis. These
findings can be detected in 70% to 90% of patients one year
after the transplant [7].
In all of these cases, however, the real recurrence rate can
only be estimated through routine serial biopsies, considering
that 20% to 30% of patients do not evolve to increased
aminotransferase levels, and that such an increase lacks
specificity, potentially resulting from other events, such as
rejection, ischemia or opportunistic infections [8]. In the Liver
Transplant Sector of the Hospital das Clínicas da Faculdade
de Medicina da Universidade de São Paulo (HCFMUSP,
University of São Paulo School of Medicine Hospital das
Clínicas), protocol biopsies are carried out every six months in
the first year after the transplant, every year between the second
and forth year after the transplant, and every three years after
the sixth year of the transplant. An evaluation of 43 patients
demonstrated histological recurrence caused by chronic
hepatitis in 80% of cases in an average period of 9.9 months [9].
Risk Factors for the Severity of Post-Transplant HCV
Recurrence of Hepatitis C
The factors that determine the evolution of hepatitis C in
patients submitted to liver transplant can be variables related
to the donor and receptor, viral factors, and events associated
with the transplant, resulting in greater severity of the disease
and higher rates of graft loss. The factors that are more
consistently associated with the severity of the disease are:
advanced age of donors, treatment for acute cellular rejection
involving pulse therapy with corticosteroids or administration
of OKT3, and infection with the cytomegalovirus [3,5,7,10]. A
better understanding of the factors that contribute to the
progression of the disease may indicate the potentially
modifiable mechanisms of its evolution.
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70
Liver Transplant and Hepatitis C Treatment
Donor and Receptor Factors
The use of older liver donors is a factor that negatively
affects the fibrosis progression rate. Recent studies show a
tendency toward a ten-year increase in the age of donors in the
last decade. This measure, although applied to compensate for
the low availability of organs, has been shown to have a direct
influence on the degree of fibrosis in recurrent hepatitis C.
Recent studies have shown that the ten-year difference in donor
age (40 versus 50 years) has been associated with greater fibrosis
progression (from 0.6 to 2.1 units a year) and with a decrease in
the interval of appearance of cirrhosis (of up to eight years).
Donor age seems to influence graft survival only in HCVpositive patients. However, there is little chance that this will
change, since very few transplant programs are able to pair
younger donors with HCV-positive receptors [11,12].
The involvement of immunogenetic factors is also
considered, with studies that observed the association
between HLA-B14 and HLA-DRB104 as beneficial to hepatitis
C evolution, and the mismatch between the donor/receptor
HLA-DRB1 with an increased recurrence risk [13,14].
Other donor factors that require further investigation
include the use of live donors, hepatic iron content, and hepatic
steatosis [15,16].
Viral Factors
Some studies have associated high viral load before or
soon after the transplant with the severity of post-transplant
HCV recurrence. An analysis involving 284 North-American
and Spanish patients showed that the pre-transplant viral load
is an independent factor in the progression of fibrosis. In
another study carried out in the United States, the five-year
survival of patients submitted to transplant for HCV was found
to be lower in patients with viral loads higher than one million
mEq/mL [7].
The importance of the HCV genotype in the progression
of the disease remains controversial. Although most of the
studies conducted in the United States failed to show this
association, a large collaborative European study showed a
higher rate of progression and severity in transplant patients
infected with HCV genotype 1b. One hypothesis is that, in
the liver transplant context, the host immune response to HCV
is stronger for the 1b genotype than for other genotypes, and
that the tissue lesion is associated with this response [17].
Factors Associated with the Transplant
The treatment of acute rejection episodes with the use of
corticosteroid pulse therapy or anti-lymphocyte preparations
has been associated with greater severity of hepatitis C
recurrence. However, for such patients, the use of
immunosuppressive regimens is recommended, which is
sufficient to prevent moderate or severe rejection, as is the
subsequent use of corticosteroid pulse therapy or OKT3
administration, but not to the point of exacerbating the
hepatitis C progression or causing other long-term
complications [5].
Considering that one of the hypotheses put forth to explain
the more severe HCV recurrence observed in recent years is
the increased potency of immunosuppressive agents, several
studies have been carried out to minimize or even abandon
the use of the immunosuppressive regimen. These studies
have shown diverging results. However, there were differences
among the studies in terms of the initial immunosuppressive
doses and the dose reduction rates. In some studies, the
prolonged use of maintenance corticosteroid therapy was
associated with lesser severity of the hepatic disease
recurrence. In this case, the method of reducing the dose was
important, and gradual reductions have been associated with
less aggressive forms of hepatitis C [18,19].
The use of azathioprine or mycophenolate mofetil has not
been shown to have any consistent effect on HCV recurrence.
Although cyclosporine has been shown to have antiviral
properties in vitro, it has not shown to have any advantage
over tacrolimus in clinical practice [20,21]. The long-term use
of sirolimus can provide some benefit, since it has antifibrotic
and potential antiviral effects, although such studies are still
preliminary and do not support its preferential use in this
group of patients. The use of new drugs, such as sirolimus
and the interleukin 2 receptor antagonist, requires controlled
and prospective studies. Therefore, the general
immunosuppression status seems to be one of the possible
determinant events in the course of recurrent hepatitis C
[20,22,23].
Another factor that is associated with the transplant and
negatively influences the post-transplant evolution of hepatitis
C is the presence of infection with the cytomegalovirus, which
leads to the worsening of fibrosis [24].
Pre- and Post-Transplant Approach to Treating HCV-Positive
Patients
Antiviral therapy is the main strategy used in treating HCVpositive patients. However, the ideal moment at which to
intervene remains unknown. The authors of most studies have
initiated the treatment for HCV recurrence when there is
histological evidence of the disease. Alternative treatments
include the use of antiviral therapy before or soon after the
transplant, when there is still no clinical evidence of recurrent
disease. This is known as pre-emptive therapy. Antiviral
therapy is generally less efficient and less well tolerated in the
transplant patient than in the immunocompetent patient.
Treatment of Patients with Cirrhosis Who are on the
Transplant Waiting List
Viral clearance in the patient with cirrhosis, in addition to
providing better expectations for the transplant (increased
graft survival), can even interrupt the progression of fibrosis
in these patients, and, in some cases, preclude the need for
the liver transplant.
Studies have proven that, although the side effect rates
are high, the rate at which a sustained virological response is
achieved in patients with compensated cirrhosis treated with
www.bjid.com.br
progressively higher doses of conventional interferon and
ribavirin is approximately 22%, and can be even higher in those
infected with genotype 2 or 3. When submitted to transplant,
such patients do not present recurrence of the disease,
confirming the validity of this therapeutic strategy [25].
In one study, carried out by Forns et al., 30 transplant
waiting list patients of different functional classes were
submitted to antiviral treatment. In that study, the efficacy of
the treatment was evaluated on the basis of the virological
response throughout the treatment period, as well as on the
rate at which a sustained virological response was achieved.
The simple reduction in the HCV viral load before the
transplant was sufficient to avoid recurrence after the
transplant (efficacy of approximately 66%). In addition, none
of the patients achieving a sustained virological response
experienced disease recurrence [26].
Most of the studies involving this population of patients
have evaluated the efficacy of conventional interferon. Overall,
they concluded that the treatment is recommended for patients
with Child-Turcotte-Pugh class A or B cirrhosis and a model
for end-stage liver disease (MELD) score lower than 18, or
even in patients with decompensated cirrhosis. However, in
the last case, following the treatment protocol in a center with
support and possibility of immediate transplant [26].
In a recent study of data collected in the HCFMUSP Liver
Transplant Sector, 37 transplant waiting list patients with HCVinduced cirrhosis were submitted to antiviral treatment. This
population was composed of 46% women and 54% men, with
a mean age of over 50 years. The predominant genotypes
were 1 and 3. There was history of ascites and hepatic
encephalopathy in 43.2% and 8.1% of the patients infected
with genotypes 1 and 3, respectively. History of varicose
digestive hemorrhage two months before the treatment was
present in 5.4% of patients, and a history of spontaneous
bacterial peritonitis was common (in 8.1%). Pegylated
interferon was used in only five patients (all infected with
genotype 1), and conventional interferon was used in the
remaining patients. Both were used in combination with
ribavirin. The mean duration of treatment was 7.9 months.
The most common side effect associated with the treatment
discontinuation was neutropenia (in 37.7%). Of the 37 patients,
14 (37.8%) presented viral load reduction of at least 2 log. In
that study, the presence of compensated or decompensated
cirrhosis did not affect the treatment response [27].
Recent studies describe the treatment with pegylated
interferon and ribavirin in patients with decompensated
cirrhosis. Although they present a considerable virological
response, the frequency of severe complications necessarily
leads to the need to always analyze the risk/benefit ratio before
the decision to initiate treatment is made, also considering the
feasibility of an emergency transplant [28,29].
Pre-Emptive Therapy
Antiviral therapy before the establishment of histologically
confirmed disease presents theoretical advantages,
71
considering that, immediately after the transplant, the HCV
viral load and the degree of hepatic fibrosis tend to be lower.
This could provide a better response to the treatment, similar
to what occurs in nontransplant patients. However, this is a
moment at which the immunosuppression is still high, interfering
with the antiviral response, and the antiviral regimen tolerability
is too low in view of all of the other post-transplant clinical
complications, such as infections and cytopenias. In addition,
the immunomodulatory effect of interferon can increase the
risk of acute cellular rejection, which is higher in this phase of
the transplant process. Another criticism of pre-emptive
treatment is that it does not distinguish patients who will actually
evolve to a more significant recurrence of the disease and for
whom treatment is indicated, from those who might have no
need of antiviral therapy after the transplant [30,31].
Controlled studies have shown that treatment with the
combination of conventional interferon and ribavirin has an
advantage over monotherapy with interferon. There was a
delay in the appearance of recurrence in those patients, who
presented viral load reduction and better histological profiles.
Studies involving the use of pegylated interferon and ribavirin
have also demonstrated histological improvement, although
their results are generally disappointing, with sustained
virological response rates of 7-13% with the use of isolated
interferon, 16-33% with interferon and ribavirin, and 9% with
isolated pegylated interferon [32].
This strategy is not applicable to all patients. Those with
better MELD scores before the transplant seem to be the best
candidates. The need to reduce the dose or even discontinue
the treatment is common, typically caused by cytopenias and
concomitant renal dysfunction, with secondary anemia, which
makes the use of ribavirin particularly difficult [33].
Post-Transplant Treatment
In general, most patients submitted to transplant for
cirrhosis caused by HCV are treated after the transplant, when
recurrence is already an established event. Unfortunately, most
studies that support this treatment strategy have been
uncontrolled, preventing the determination of the treatment
risks, acute/chronic rejection rates, and even the evaluation
of the therapeutic efficacy.
This is a population that, in principle, presents the worst
prognostic factors of evolution and treatment response, since
it comprises older patients who are infected with genotype 1,
have high viral loads and present more extensive fibrosis, as
well as more often having a history of previous treatment.
These characteristics are also associated with the fact that
transplant patients present comorbidities that frequently
prevent the use of full-dose therapies [34].
In this group of patients, protocol biopsies are an essential
means of assessing the degree of hepatic fibrosis and should
be carried out whenever clinically indicated (by an increase in
aminotransferase levels) or at least on an annual basis, with
the specific purpose of detecting and monitoring HCV
recurrence [35].
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72
The results of this group are no less disappointing, with a
sustained virological response rate of 12.5% with isolated
interferon, 21% with interferon and ribavirin, and 9% with
isolated pegylated interferon. In uncontrolled studies the
combined use of pegylated interferon and ribavirin proved
to be the best strategy, with responses between 30% and
45% [2,36-38].
The optimal duration of antiviral therapy remains
undefined. Although most recent studies established
treatment periods of 48 to 52 weeks, the validity of
prolonging treatment in patients who achieved virological
response by the end of the standard treatment period is still
in question [39-41].
The advantages of therapy that begins within 6 to 24
months after the transplant, compared with pre-emptive
therapy, is that these patients require less
immunosuppression, present better clinical status, and are
at lower risk of acute or chronic rejection [7].
The occurrence of acute or chronic rejection has not
been a limiting factor to the treatment, although there are
some reports on this subject [42].
The use of ribavirin as isolated therapy or as maintenance
after the combined use with interferon has no subside in the
literature [43].
Use of Adjuvant Therapy
In this group of patients, one of the central issues is the
high rate of side effects from antiviral drugs, especially
cytopenia, which often requires dose reduction or even
discontinuation of the treatment. The risk/benefit ratio of the
use of erythropoietin or granulocyte colony-stimulating factor
has not been well established. However, its use has facilitated
the maintenance of antiviral treatment and the use of optimal
doses of ribavirin and interferon [44]. Efforts have been made
to investigate the use of ribavirin substitutes that do not cause
hemolysis, such as viramidine, although controlled studies
are still needed in order to determine the best strategy in
relation to the adverse effects of the therapy [5].
Second Transplants in Cases of Hepatitis C Recurrence
Although a second transplant is always an option in
patients presenting hepatitis C recurrence, this strategy is
historically associated with disappointing results. Receptor
age, total bilirubin, high prothrombin time, older donor age,
admission to the intensive care unit, high creatinine level,
and high MELD score are predictive factors of short survival
after a new transplant. Second transplants remain
controversial and require comprehensive discussions in view
of the low availability of organs and the use of MELD score
as an organ allocation criterion, which implies that second
transplants will be given to recurrent patients presenting
more severe clinical profiles. In general, a second transplant
is recommended if one of the variables related to recurrence,
and thus the natural history of HCV recurrence, can be altered
[45-47].
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mismatch affects the outcome of hepatitis C disease
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15. Imber C.J., St.Peter S.D., Handa A., Friend P. Hepatic steatosis
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and metabolic complication: results from a prospective
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20. Berenguer M., Aguilera V., Prieto M., et al. Effect of calcineurin
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21. Hilgard P., Kahraman A., Lehmann N., et al. Cyclosporine versus
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22. Jain A., Kashyap R., Demetris A.J., et al. A prospective randomized
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liver fibrosis in patients with chronic hepatitis C after orthotopic
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26. Forns X., Garcia-Retortillo M., Serrano T., et al. Antiviral therapy
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35. Sebagh M., Rifai K., Féray C., et al. All liver recipients benefit
from the protocol 10-year liver biopsies. Hepatology
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36. Wang C.S., Ko H.H., Yoshida E.M., et al. Interferon-based
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37. Murkherjee S., Lyden E. Impact of pegylated interferon alpha-2b
and ribavirin on hepatic fibrosis in liver transplant patients
with recurrent hepatitis C: an open-label series. Liver Int
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38. Heydtmann M., Freshwater D., Dudley T., et al. Pegylated
interferon alpha-2b for patients with HCV recurrence and graft
fibrosis following liver transplantation. Am J Transplant
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39. Lavezzo B., Franchello A., Smedile A., et al. Treatment of recurrent
hepatitis C in liver transplants: efficacy of a six versus a twelve
month course of interferon alfa-2b with ribavirin. J Hepatol
2002;37:247-52.
40. Rodriguez-Luna H., Khatib A., Sharma P., et al. Treatment of
recurrent hepatitis C infection after liver transplantation
with combination of pegylated interferon alpha-2b and
ribavirin: an open-label series. Transplantation
2004:77:190-4.
41. Bizzolon T., Ahmed S.N.S., Radenne S., et al. Long term histological
improvement and clearance of intrahepatic hepatitis C virus
RNA following sustained response to interferon-ribavirin
combination therapy in liver transplanted patients with hepatitis
C recurrence. Gut 2003;52:283-7.
42. Khalili M., Vardanian A.J., Hamerski C.M., et al. Management of
hepatitis C-infected liver transplant recipients at large North
American centres: changes in recent years. Clin Transplant
2006;20:1-9.
43. Schiano T.D., Martin P. Management of HCV infection and liver
transplantation. Int J Med Sci 2006;3:79-83.
44. Gotardo D.R.M., Abdala E., Bonazzi P.R., et al. Safety of recurrent
hepatitis C treatment after liver transplantation with use of
adjuvants. Liver Transplant 2007;13(suppl 1):S152.
45. McCashland T.M. Retransplantation for recurrent hepatitis C:
positive aspects. Liver Transplant 2003;9(suppl 3.):S67-S72.
46. Wall W.J., Khakhar A. Retransplantation for recurrent hepatitis C:
the argument against. Liver Transplant 2003;9(suppl 3):S73-S8.
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74
Adverse Event Management
Aline Gonzalez Vigani
Department of Viral Hepatitis Studies, State University of Campinas (Unicamp); Campinas, SP, Brazil
The treatment of hepatitis C involves combining
conventional interferon (IFN) or pegylated IFN (PEG-IFN) with
ribavirin (RBV). These therapeutic regimens are associated
with numerous adverse events, among which constitutional
and neuropsychiatric symptoms, as well as hematological
abnormalities, stand out [1,2]. The adverse events observed
with the use of PEG-IFN or conventional IFN are similar, and
the frequencies of those events are shown in Table 1 [3-5].
Adverse events resulting from the treatment of hepatitis C
can jeopardize the quality of life of patients and their response
to treatment. The control of those events involves medicinal
and non-medicinal interventions. The latter include a
reduction in the dosage of IFN or RBV and discontinuation of
the treatment.
A temporary or permanent reduction in the dosage of PEGIFN, conventional IFN or RBV as a result of an adverse event
is necessary in approximately 30% of patients. In 10% of
patients it is necessary to discontinue the treatment [4].
Hematological abnormalities (neutropenia, anemia and
thrombocytopenia) and depression are the most common
causes of dose reduction.
The maintenance of the dosages of medications used in
the treatment of hepatitis C and of the recommended course
of treatment are important for the effectiveness of the therapy,
Table 1. Adverse events associated with using the pegylated
interferon-ribavirin combination in the treatment of hepatitis C
Adverse event
Reaction at the site of injection
Fatigue
Headache
Myalgia
Fever
Chills
Alopeica
Artralgia
Irritability
Depression
Anorexia
Dermatitis
Anemia
Neutropenia
Thrombocytopenia
Approximate
incidence (%)
36-58
50-64
50-62
42-56
43-56
24-48
28-36
27-35
24-35
21-34
14-32
16-21
12-22
17-20
3-6
as shown in Figure 1 [6]. Dose reduction is associated with
lowering the rate at which a sustained viral response (SVR) is
achieved. Early identification and strategies for controlling
adverse events are important in the prevention of moderate
and severe complications. These practices also attenuate the
deleterious effects on the quality of life of patients and maximize
the effectiveness of treatment for hepatitis C.
Constitutional Symptoms
Constitutional symptoms such as fatigue, headache and
myalgia are the most common adverse events in patients
Figure 1. Effect of dose reduction of pegylated interferon
(PEG-IFN) and ribavirin (RBV) in relation to the sustained
viral response (SVR) rate.
treated with the combination of IFN and RBV (Table 1).
Practically all patients manifest at least one such event during
the administration of the initial doses of IFN. These symptoms
typically disappear or become less intense after the first month
of treatment. Paracetamol, acetaminophen and ibuprofen taken
immediately before the injection of IFN alleviate the symptoms.
Adequate hydration, together with light to moderate exercise,
can help minimize these side effects.
Hematological Effects
In approximately 25% of patients, it is necessary at least
to reduce the dosage of IFN or RBV due to abnormalities in
laboratory test results [4]. Conventional IFN and PEG-IFN
have both been associated with the suppression of
hematopoiesis. In addition, hemolytic anemia occurs in all
patients treated with RBV, although the intensity of that anemia
is variable.
2007;11 (5) Suppl. 1:74-78.
Anemia
Anemia resulting from the treatment of hepatitis C is a
multifactorial side effect. Those factors increase the
destruction of red blood cells and reduce their production.
The increased destruction of red blood cells occurs due to
hemolysis triggered by RBV, and the reduction in red blood
www.bjid.com.br
cell production occurs due to IFN-induced suppression of
bone marrow erythroid precursors [7,8].
The RBV penetrates erythrocytes, in which
monophosphate, diphosphate and triphosphate are
phosphorylated. Those pharmacologically active forms are
incapable of passing through the erythrocyte membrane, and
so remain intracellularly retained at a concentration 60 times
greater than that of plasma [7,8]. The accumulated
phosphorylated derivates are slowly eliminated from the
erythrocytes, which have a half-life of 40 days. In contrast,
the elimination of RBV from plasma is rapid, since it has a halflife of 24 hours.
The mechanisms by which IFN can exacerbate RBVinduced anemia include suppression of hematopoiesis in bone
marrow and an increase in the destruction of erythroid
precursor cells [9].
Anemia starts and develops almost immediately after
the initiation of therapy, becoming more intense after 4 to 6
weeks of treatment. The average decrease in the level of
hemoglobin (Hb) is 2.5 to 3.0 g/dL, but more intense degrees
of hemolysis can occur. Anemia associated with combined
therapy can exacerbate other side effects resulting from
the treatment of hepatitis C such as dyspnea, fatigue,
dizziness and headache.
Non-pharmacological control of the treatment of anemia
involves dose reduction or permanent discontinuation of RBV
[9]. In patients without heart disease, a reduction in the dosage
of ribavirin when the levels of Hb fall below 10 g/dL is
recommended, and discontinuation is recommended when Hb
levels are under 8.5 g/dL (Table 2) [10]. Those
recommendations are more restricted to patients with stable
heart disease. In rare cases, when the level of Hb is lower than
8.5 g/dL, a transfusion of packed red blood cells, in
conjunction with discontinuation of the treatment for hepatitis
C, might be necessary.
Between 9 and 22% of patients receiving combined
treatment for hepatitis C require an RBV dose reduction due
to anemia [3,4].
However, the strategy of RBV dose reduction has been
associated with a modest increase in the level of Hb (˜1 g/dL)
at 4-8 weeks after the reduction, as well as with a decrease in
the rate at which an SVR is achieved (Figure 1) [6,11].
Hematopoietic growth factors such as alpha erythropoietin
can be an alternative in relation to RBV dose reduction for the
treatment of anemia arising during the treatment of hepatitis C.
Alpha erythropoietin is a synthetic glycoprotein that has a
mechanism of action similar to that of endogenous erythropoietin,
which is a hormone produced by the renal peritubular capillaries
that stimulates erythropoiesis in bone marrow.
Studies indicate that therapy with erythropoietin at the
dosage of 40,000 IU once a week elevates the level of Hb after
one week of use and thus permits the maintenance of RBV
dosage in patients that become anemic during combined
therapy for hepatitis C [12,13]. In the study conducted by
Afdhal et al., the initial dose of RBV was maintained in 88% of
75
the patients who received erythropoietin during treatment for
hepatitis C, and Hb levels increased (from 10.6 ± 0.9 to 13.2 ±
1.2 g/dL) in those same patients [13]. The initial dose of RBV
was maintained in 66% of the patients who received a placebo.
The cost of alpha erythropoietin is high, but it is a therapeutic
option for patients who develop an Hb level < 10 g/dL during
treatment of hepatitis C [14].
Neutropenia
A decrease in neutrophil counts to below normal values
occurs in the majority of patients receiving conventional IFN
or PEG-IFN and results from the suppression of bone marrow
caused by those medications [15]. Neutropenia (neutrophil
count <750 cells/mL) is more common with the use of PEGIFN than with that of conventional IFN, with values of 20%
and 4%, respectively [3,4]. Intense neutropenia (<500 cells/
mm3) is also more common in patients treated with PEG-IFN
than in those treated with conventional IFN, with values of
5% and 1%, respectively.
After a single injection of PEG-IFN, neutrophil counts
decrease, on average, 21% within the first 24 hours but
generally stabilize at higher levels thereafter. Therefore, the
measurement of neutrophil counts should be carried out before
the administration of IFN so that more reliable values can be
obtained.
In patients who develop neutropenia during treatment for
hepatitis C, in general, IFN dose reduction or discontinuation
of treatment is necessary. Recommendations for dose
reduction or discontinuation of the treatment due to
neutropenia are shown in Table 2. Neutrophil counts generally
return to pretreatment levels 2 to 4 weeks after the end of
treatment [2].
The clinical implications of IFN-related neutropenia are
not associated with an increased risk of infectious
complications [16,17]. The low infection rates observed in
neutropenic patients undergoing treatment for hepatitis C can
reflect an early reduction in IFN dosage and, in addition, can
indicate that those patients have a lower risk of infection when
compared with neutropenic patients undergoing
chemotherapy.
Filgrastim is beginning to be used as an adjuvant therapy,
at the dose of 300 µg 1 to 3 times a week, to increase neutrophil
counts in patients who have less than 750 cells/mm3 during
treatment for hepatitis C. However, clinical experience with
filgrastim in such cases is still limited [10,15]. Filgrastim is
a granulocyte-colony stimulating factor structurally similar
to that produced by human cells and obtained through
genetic engineering. Filgrastim interacts with receptors on
the surface of myeloid progenitor cells in bone marrow,
inducing proliferation, differentiation and activation of
functional cells.
Thrombocytopenia
Thrombocytopenia observed in patients undergoing
treatment for hepatitis C is a consequence of IFN-induced
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76
Table 2. Dose modifications for adverse events resulting from therapy with interferon and ribavirin according to manufacturer
recommendations
Hematological parameters
Hemoglobina
<10 g/dL
<8.5 g/dL
Platelets
Neutrophils
<25,000/mm3
<750/mm3
<500/mm3
PEG-IFN-α
α 2b
PEG-IFN-α
α 2a
IFN
RBV
Reduce to 11 mg/kg
Discontinue
Discontinue
Reduce to
1 μg/kg
Discontinue
Discontinue
Reduce to
135 μg
Discontinue
Discontinue
Reduce by 50%
Discontinue
Discontinue
PEG-IFN=pegylated interferon; RBV=ribavirin. aIn patients without heart disease or in patients with a history of stable heart disease
undergoing combined therapy, IFN dosage should be reduced by 50% and RBV dosage should be lowered to 200 mg/day if there is a decrease
in hemoglobin of ≥2 g/dL within a period of 4 weeks. Both medications should be permanently discontinued if hemoglobin drops to < 12
g/dL after dose reduction.
Figure. 2. Algorithm for the pharmacological treatment of INF-induced depression.
BDI=Beck Depression Inventory; CES-D=Center for Epidemiological Studies Depression Rating Scale; Zung SDS=Zung Self Rating Depression Scale.
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77
bone marrow suppression. A drop in platelet counts during
combined treatment for hepatitis C is common, but IFN dose
reduction or discontinuation of treatment as a result of that
drop is uncommon [2,3]. Thrombocytopenia requiring IFN
dose reduction is more common in patients treated with PEGIFN and RBV than in those treated with conventional IFN and
RBV, with frequency rates of 4% and 1%, respectively [18].
Recommendations for the discontinuation of PEG-IFN or
conventional IFN as a result of thrombocytopenia are shown
in Table 2. Platelet counts generally return to pretreatment
levels by 4 weeks after the discontinuation of treatment [2].
There are ongoing studies that aim to evaluate the effectiveness
and safety of growth factors that stimulate the production
and maturation of megakaryocytes in thrombocytopenic
patients undergoing treatment for hepatitis C.
Rare cases of autoimmune thrombocytopenic purpura have
been described during the treatment for hepatitis C, and the
presence of that disorder should be investigated in rare cases
involving an intense decrease in platelet counts.
Thyroid Disease
Approximately 1-6% of individuals treated with IFN
develop thyroid abnormalities [23]. In all patients, an
evaluation of thyroid function is recommended. Levels of
thyroid-stimulating hormone should be determined before the
initiation of treatment for hepatitis C, every 12 weeks during
treatment and once after the end of the treatment. Individuals
who develop hypothyroidism while undergoing treatment
should receive hormonal replacement therapy.
Neuropsychiatric Symptoms
Approximately 20% to 30% of patients report depressive
symptoms during treatment for hepatitis C [19]. The control of
depression in patients who are candidates to receive treatment
for hepatitis C is shown in Figure 2 [20]. In the pretreatment
phase, it is important to establish the psychiatric status of the
patient, which involves determining whether there is a history
of depression, assessing the mood state and identifying abuse
of illegal substances. Patients who abuse drugs or who have a
complex psychiatric history involving disorders such as
schizophrenia and bipolar disorder or who are receiving
psychiatric treatment should be jointly accompanied by a
psychiatrist during treatment for hepatitis C. Patients with
current or previous depression should be evaluated before the
initiation of treatment for hepatitis C, preferably through the
use of scales that infer the intensity of depression. In patients
with moderate depression, antidepressant medication can be
given 4 weeks before the initiation of treatment for hepatitis C.
Antidepressants belonging to a class of selective
serotonin reuptake inhibitors (SSRIs) constitute the treatment
of choice for IFN-associated depression [19]. Such
antidepressants are safe and well-tolerated in patients with
hepatic disease. In addition, the treatment success rate in
patients with depression during treatment for hepatitis C is
near 90% [18]. Fluoxetine, sertraline, citalopram, paroxetine
and other SSRIs can have a slightly sedative effect, and the
choice of SSRI to be used should be based on that effect and
on the predominant symptomatology of the patient [21]. In
patients with fatigue or cognitive slowness, fluoxetine or
sertraline can be preferred over paroxetine, which tends to be
less stimulating. In case of suicidal ideation or attempted
suicide, the treatment of hepatitis C should be immediately
suspended [22]. The dose of antidepressant medication started
during treatment of hepatitis C should be slowly reduced over
a 6 to 12 month period after the end of the treatment.
Retinopathy
The use of IFN can trigger or aggravate prior retinopathy.
Subconjunctival hemorrhage and retinal hemorrhage have
been reported during treatment with IFN [19]. Patients with
risk factors for retinopathy such as systemic arterial
hypertension and diabetes mellitus should undergo
ophthalmological examination before and during therapy under
the supervision of an ophthalmologist. Treatment should be
discontinued in individuals who present either retinal lesions
during treatment or the worsening of a prior lesion.
Pulmonary Side Effects
Dry cough, which can occur during treatment, has been
associated with the use of RBV. In most cases the cough is
tolerable, but occasionally it is necessary to discontinue the
use of RBV. Cases in which cough becomes productive or is
accompanied by abnormal pulmonary auscultation findings
or fever, pneumonia should be investigated. Interstitial
pneumonia can be severe, but it is normally reversible with
the discontinuation of therapy [19].
References
1. Maddrey W.C. Safety of combination interferon alfa-2b/ribavirin
therapy in chronic hepatitis C-relapsed and treatment-naive
patients. Semin Liver Dis 1999;19 Suppl 1:67-75.
2. Fried M.W. Side effects of therapy of hepatitis C and their
management. Hepatology 2002;36(5 Suppl 1):S237-44.
3. Manns M.P., McHutchison J.G., Gordon S.C., et al. Peginterferon
alfa-2b plus ribavirin compared with interferon alfa-2b plus
ribavirin for initial treatment of chronic hepatitis C: a
randomised trial. Lancet 2001;22:958-65.
4. Fried M.W., Hadziyannis S.J. Treatment of chronic hepatitis C
infection with peginterferons plus ribavirin. Semin Liver Dis
2004;24 Suppl 2:47-54.
5. Hadziyannis S.J., Sette H. Jr., Morgan T.R., et al. Peginterferonalpha2a and ribavirin combination therapy in chronic hepatitis
C: a randomized study of treatment duration and ribavirin dose.
Ann Intern Med 2004;2;140(5):346-55.
6. Shiffman M.L., Di Bisceglie A.M., Lindsay K.L., et al. Hepatitis C
Antiviral Long-Term Treatment Against Cirrhosis Trial Group.
Peginterferon alfa-2a and ribavirin in patients with chronic
hepatitis C who have failed prior treatment. Gastroenterology
2004;126(4):1015-23.
7. Bodenheimer H.C. Jr., Lindsay K.L., Davis G.L., et al. Tolerance
and efficacy of oral ribavirin treatment of chronic hepatitis C:
a multicenter trial. Hepatology 1997;26(2):473-7.
8. De Franceschi L., Fattovich G., Turrini F., et al. Hemolytic anemia
induced by ribavirin therapy in patients with chronic hepatitis
C virus infection: role of membrane oxidative damage.
Hepatology 2000;31(4):997-1004.
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78
9. Kowdley K.V. Hematologic side effects of interferon and ribavirin
therapy. J Clin Gastroenterol 2005;39(1 Suppl):S3-8.
10. Afdhal N.H. Role of epoetin alfa in maintaining ribavirin dose.
Gastroenterol Clin North Am 2004;33(1 Suppl):S25-35.
11. Sulkowski M.S., Wasserman R., Brooks L., et al. Changes in
haemoglobin during interferon alpha-2b plus ribavirin
combination therapy for chronic hepatitis C virus infection. J
Viral Hepat 2004;11(3):243-50.
12. Dieterich D.T., Wasserman R., Brau N., et al. Once-weekly epoetin
alfa improves anemia and facilitates maintenance of ribavirin
dosing in hepatitis C virus-infected patients receiving ribavirin
plus interferon alfa. Am J Gastroenterol 2003;98(11):2491-9.
13. Afdhal N.H., Dieterich D.T., Pockros P.J., et al. Epoetin alfa
maintains ribavirin dose in HCV-infected patients: a prospective,
double-blind, randomized controlled study. Gastroenterology
2004;126(5):1302-11.
14. Devine E.B., Kowdley K.V., Veenstra D.L., Sullivan S.D.
Management strategies for ribavirin-induced hemolytic anemia
in the treatment of hepatitis C: clinical and economic
implications. Value Health 2001;4(5):376-84.
15. Collantes R.S., Younossi Z.M. The use of growth factors to manage
the hematologic side effects of PEG-interferon alfa and ribavirin.
J Clin Gastroenterol 2005;39(1 Suppl):S9-13.
16. Sulkowski M.S. Management of the hematologic complications
of hepatitis C therapy. Clin Liver Dis 2005;9(4):601-16.
17. Soza A., Everhart J.E., Ghany M.G., et al. Neutropenia during
combination therapy of interferon alfa and ribavirin for chronic
18. Aspinall R.J., Pockros P.J. The management of side-effects during
therapy for hepatitis C. Aliment Pharmacol Ther
2004;20(9):917-29.
19. Russo M.W., Fried M.W. Side effects of therapy for chronic hepatitis
C. Gastroenterology 2003;124(6):1711-9.
20. Raison C.L., Demetrashvili M., Capuron L., Miller A.H.
Neuropsychiatric adverse effects of interferon-alpha:
recognition and management. CNS Drugs 2005;19(2):105-23.
21. Edwards J.G., Anderson I. Systematic review and guide to selection
of selective serotonin reuptake inhibitors. Drugs
1999;57(4):507-33.
22. Janssen H.L., Brouwer J.T., van der Mast R.C., Schalm S.W. Suicide
associated with alfa-interferon therapy for chronic viral
hepatitis. J Hepatol 1994;21(2):241-3.
23. Huang M.J., Tsai S.L., Huang B.Y., et al. Prevalence and significance
of thyroid autoantibodies in patients with chronic hepatitis C
virus infection: a prospective controlled study. Clin Endocrinol
(Oxf) 1999;50(4):503-9.
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79
Treatment Options in the Management of Thrombocytopenia in Patients Infected with HCV
André Cosme de Oliveira
Liver Transplant Section of the Clinical Hospital of University of São Paulo School of Medicine (HC-FMUSP); São Paulo, SP, Brazil
Thrombocytopenia (platelet count < 80,000/mm3) in
patients with chronic hepatitis has been associated with two
factors: the first is hypersplenism resulting from splenomegaly
in portal hypertension [1]. The spleen continuously
sequesters one-third of circulating platelets, so that
splenomegaly increases the fraction of platelets trapped in
the splenic sinusoids, especially when resulting from passive
congestion or an increase in venous portal pressure [1].
Hypersplenism seems to be the most common cause of
thrombocytopenia associated with liver cirrhosis and portal
hypertension.
The second mechanism is related to the decreased
production of thrombopoietin, a hormone produced by
hepatocytes, which regulates the development of the
megakaryocyte. In cirrhosis, due to the reduction in the mass
of functioning hepatocytes, there can be a reduction of
thrombopoiesis in the bone marrow, leading to
thrombocytopenia in the peripheral blood [1].
In some situations, patients who are otherwise eligible for
HCV treatment with interferon and ribavirin cannot be so
treated because their platelets counts are low, which
jeopardizes the treatment [2,3].
Nevertheless, hepatitis C patients treated with interferon and
ribavirin also present a drop in the platelet count as a side
effect [2,3].
There is as yet no treatment consensus in the literature for
the management of these patients. We therefore present some
related studies that address the management of these patients,
in the pre-treatment and intra-treatment phases.
Patients receiving interferon alpha or peginterferon alpha
can present a 30-50% reduction in the baseline platelet count,
and a dose reduction is necessary in approximately 4% of the
patients [2,3]. We should consider possible reduction of the
dose when platelet counts drop to < 50,000/mm3, and
discontinuation of treatment when < 25,000/mm3.
When using peginterferon alpha-2, the weekly dose can
be reduced from 180 μg to 135 μg, or even to 90 μg. While
using peginterferon alpha-2b, the weekly dose can be reduced
from 1.5 μg/kg to 1.0 μg/kg, or even to 0.5 μg/kg [2,3].
Human recombinant interleukin (IL)-11 (oprelvekin,
Neumega®; Wyeth Laboratories, Collegeville, PA, USA) can
be used as a way to stimulate the increase in the number of
platelets at a dose of 5 μg/kg/day, subcutaneously, initially
for 7 days. If necessary, maintenance can consist of 1 to 3
doses per week during HCV drug therapy, similarly to what is
recommended for filgrastim (granulocyte colony-stimulating
factor) [4].
2007;11 (5) Suppl. 1:79-80.
Administration of IL-11 has multiple effects on the
hematopoietic system, specifically promoting the proliferation
and maturation of megakaryocytes and inducement of
neutrophilia and thrombocytosis [5].
In patients with cirrhosis, its use should be restricted to
those with Child-Pugh class A or B cirrhosis [5].
In a study conducted by Ghalib et al. [6], none of the
patients presented increased platelet counts until day 4 of
daily use of the medication, and the maximal therapeutic
response was observed near day 13 (Figure 1). At 6 to 8 days
after the initiation of treatment, 89% of the patients presented
duplication of the initial number of platelets, and 78% of the
patients presented platelet counts > 80,000/mm3.
Another treatment option for thrombocytopenia in these
patients is splenic embolization.
Figure 1. Ratio between platelet counts and duration of
treatment with interleukin-11.
Splenic embolization should preferably be performed with
injection of polyvinyl alcohol particles between 355 and 500
µm (Trufill PVA Embolization Particles; Cordis Corp., Johnson
& Johnson Co., Miami Lakes, FL, USA) into the splenic artery
and should not exceed an embolization volume of 30% of the
initial spleen volume [7].
The most severe complications after embolization can be
ascites, thrombosis of the splenic vein or portal vein,
spontaneous bacterial peritonitis, and splenic abscess.
Abdominal pain and fever are typically present postembolization [8].
Splenic embolization presented 50% of complication in
embolizations < 30% of splenic volume, whereas, in
embolizations > 30%, 100% of the patients presented
complications [9].
Abdominal computed tomography scans can be performed
2 to 3 weeks after embolization for assessment of effectiveness
of the procedure and to identify complications [10].
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80
Thrombocytopenia in Patients Infected with HCV
All patients should be previously vaccinated against
pneumococci before embolization. In addition, antibiotic
therapy should be initiated and maintained during
hospitalization of the patient. We suggest the use of cefotaxime
and ampicillin in these cases [11]. The combined treatment
with interferon and ribavirin should not be initiated or reinitiated until 8 weeks after splenic embolization [12].
The use of human recombinant interleukin-11 (IL-11)
(oprelvekin) and splenic embolization are both feasible
treatments for these patients, taking into account the
characteristics of the individual health care facilities at which
the follow-up treatment is administered.
References
1. Moreno A., Bárcena R., Blázquez J., et al. Thrombocytopenia can
be found in patients with chronic hepatitis related to hepatitis C
virus (HCV). Both hypersplenism and decreased liver production
of thrombopoietin (TPO) have been hypothesized as
mechanisms responsible for thrombocytopenia. Hospital Ramón
y Cajal, Madrid, Spain.
2. Manns M.P., et al. Peginterferon alfa-2b plus ribavirin compared
with interferon alfa-2b plus ribavirin for initial treatment of chronic
hepatitis C: a randomised trial. Lancet 2001;358:958-65.
3. Fried M.W. Peginterferon alfa-2a plus ribavirin for chronic hepatitis
4. Hennepin County Medical Center. Management of HCV and
treatment side effects. April 10, 2005.
5. Lawitz E., Hepburn M., Casey T. A Pilot Study of Interleukin11 in Subjects with Chronic Hepatitis C and Advanced Liver
Disease
Nonresponsive
to
Antiviral
Therapy.
Gastroenterology Service; Infectious Diseases Service;
Pathology Department; Brooke Army Medical Center, San
Antonio, Texas.
6. Ghalib R., Levine C., Hassan M., et al. Recombinant Human
Interleukin-11 Improves Thrombocytopenia in Patients With
Cirrhosis. Hepatology 2003:1165-71.
7. Zhu K., Meng X., Li Z., et al. Partial splenic embolization using
polyvinyl alcohol particles for hypersplenism in cirrhosis: A
prospective randomized study. Eur J Radiol 2007;24.
8. Lee C.M., Leung T.K., Wang H.J., et al. Evaluation of the effect
of partial splenic embolization on platelet values for liver
cirrhosis patients with thrombocytopenia. World J
9. Hayashi H., Beppu T., Masuda T., et al. Predictive factors for
platelet increase after partial splenic embolization in liver
cirrhosis patients. J Gastroenterol Hepatol 2007;7.
10. Lee C.M., Leung T.K., Wang H.J., et al. Evaluation of the effect
of partial splenic embolization on platelet values for liver
cirrhosis patients with thrombocytopenia. World J
11. Foruny J.R., Blázquez J., Moreno A., et al. Safe use of pegylated
interferon/ribavirin in hepatitis C virus cirrhotic patients with
hypersplenism after partial splenic embolization. Eur J
Gastroenterol Hepatol 2005;17(11):1157-64.
12. Pålsson B., Verbaan H. Partial splenic embolization as pretreatment
for antiviral therapy in hepatitis C virus infection. Eur J
Gastroenterol Hepatol 2005;17(11):1153-5.
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81
Therapeutic Perspectives for Hepatitis C
OVERVIEW
NEW THERAPIES FOR HEPATITIS C
Jean-Michel Pawlotsky
French National Reference Center for Viral Hepatitis B, C and Delta; Department of Virology,
and INSERM U841, Hôpital Henri Mondor, Université Paris 12, Créteil, France
Overview
Hepatitis C virus (HCV) is represented by a single open
reading frame of single-stranded RNA comprising
approximately 3000 AA, positioned between 3’ and 5’
noncoding extremities. Transcription from the 5’ end results
in a compound polyprotein, generated by structural proteins
in the amino-terminal region extremity and by nonstructural
proteins in the opposing terminal region. Post-transcription
cleavage by viral and host cell proteases generates 10
individual HCV proteins [1]. The HCV cDNA clone was first
described in 1989. Subsequently, counter to the expectations
of an enormous breakthrough, there was a hiatus in the growth
of knowledge regarding the molecular virology of this virus.
Only in 1997 was the first functioning and complete HCV cDNA
clone described and used in animal models (chimpanzees) for
preliminary analyses of the viral expression using
biotechnological tools. A milestone in the study of viral
molecular biology was the description, in 1999, of a viral culture
model in tumor cells (Huh 7) using a HCV 1b subgenomic
replicon. Based on this model, despite strict limitations, other
viral fragments were used in order to evaluate the dynamics
of viral replication in different settings and with exposure to
antiviral agents. Nevertheless, the inability to obtain a complete
HCV sequence remains an obstacle to be overcome [1].
One characteristic of HCV is that is has minimal cytopathic
effects, with immunomediated hepatotropic injury. However,
it is also found in extrahepatic sites, where it has a short halflife (approximately 3 hours) and high serum turnover [2].
Standard treatment involves the administration of pegylated
interferon alpha together with ribavirin. This treatment regimen
has had modest success, a little over 50% [3], reaching 71% in
cases of dose optimization and full compliance [4]. However,
it could be much less, if we consider real life data, as well as
characteristics of the host (race, comorbidities, immune state),
of the liver disease (level of fibrosis and steatosis), of the
medication (dose, interferon type) and of the virus itself. In
addition, it is well known that genotype 1 has a lower response
rate, and certain viral proteins can subvert the stimulus induced
by interferon and ribavirin [3]. Viral kinetics, the evaluation of
the decay pattern of the viremia – in terms of intensity and
2007;11 (5) Suppl. 1:81-87.
speed – after the initiation of the therapy, has proven to be a
useful tool in the management of patients. It demonstrates
the interaction among virus, drug, and host, in a relatively
simple way: intense, rapid decay indicates a favorable case;
insignificant, slow decay indicates cases that are unfavorable,
moderate or intermediate. Such kinetic patterns allow
patients to be characterized as rapid responders, slow
responders, or nonresponders. This approach allows the
treatment to be individualized, with ideal doses per weight
and shorter, standard, or extended duration. In addition, it
allows early prediction of treatment response. Therefore,
individualized therapy constitutes the best therapeutic tool
at the moment [5].
Of course, for the sake of effectiveness, safety, and
tolerability, the current therapy is insufficient to confront the
present hepatitis C epidemic. Therefore, from a therapy with
poorly understood mechanisms of action that function
through essentially indirect means [6], we have evolved to
the specifically targeted antiviral therapy for hepatitis C [7],
that is, the search for compounds that have a direct effect on
the HCV life cycle. However, the preliminary results of current
clinical studies have demonstrated worrisome aspects
regarding safety, tolerability, and efficacy for some of these
compounds [7], which is why we believe that treatment with
interferon alpha will continue to lead the field for many years
to come, and studies involving combinations of new
compounds including interferon and, occasionally, ribavirin
are already underway. A good example of this fact are the
preliminary results of the PROVE-1 study recently presented
in Barcelona [8], in which the group that used the combination
of pegylated interferon alpha-2a 180 μg + ribavirin 1-1.2 g +
telaprevir 750 mg every 8 hours, in an intention-to-treat
analysis, showed a markedly greater response than that
observed for the control group (receiving pegylated interferon
+ ribavirin), despite the fact that the subjects were infected
with genotype 1. At week 4, 79% presented viremia < 10 IU/
mL. At week 12, 70% presented viremia < 10 IU/mL, whereas
39% presented viremia ≥ 10 IU/mL, emphasizing the loss of
cases and the intention-to-treat analysis. Nevertheless, the
incidence of rash, gastrointestinal effects, and anemia was
significantly higher in the telaprevir group.
The new therapeutic options are divided into ‘smallmolecule’ (protease and viral polymerase inhibitors and
www.bjid.com.br
82
protein inhibitors of the host – glucosidase) [9] – data
summarized in Table 1 – and drugs whose targets are still
indirect. In this second group, there are several that are
currently in Phase I trials, specifically the Toll-like receptor
agonists 7 and 9 (ANA 245 – Isatoribine – ANA 975 and
Actilon) [10] whose mechanism of immune stimulation is
broader than that induced by interferon alpha. The modulation
of the inflammation and apoptosis by caspase inhibition is
also under evaluation, and preliminary results demonstrate
that aspartate aminotransferase normalizes in the patients who
received it during trials ranging from Phase II to IDN-6556
[11]. There are other forms of interferon in phase I and II trials:
albuferon (interferon alpha-2b fused to albumin, with more
favorable posology regimen and similar efficacy to that of
pegylated interferon); omega interferon; gamma interferon;
interferon for oral use; and oral interferon inducers
(resiquimod and imiquimod). In addition, there are nucleoside
analogs similar to ribavirin (viramidine, now known as
taribavirin) in Phase III, inosine monophosphate
dehydrogenase inhibitors– VX98-497 (merimepodib) in Phase
II for previous nonresponders [12], mycophenolate mofetil,
also in Phase II for nonresponders, broad spectrum antiviral
agents in Phase II (amantadine and rimantadine) and other
immunomodulators in Phases I to III (histamine, thymosin
alpha-1, IL-10 and IL-12). Finally, therapeutic vaccines are
being studied in pre-clinical phases (E1/E2) I or II (E1, NS3NS4-NS5-core, IC41) [10].
After a brief review of developing drugs, we should bear
in mind the problems and disappointments already
encountered regarding the resistance developed in vitro or in
vivo, in addition to the already mentioned weaknesses related
to posology tolerance and complexity. Posology regimens, as
already described for HIV, can be facilitated by the association
with the protease inhibitor ritonavir, acting as a potent
inhibitor of the enzyme system function of cytochrome P450,
which would enable, in preliminary analyses, simplified
posology regimens for some of the small molecules [13].
Regarding other problems, further review will bring up some
relevant issues, in addition to the already evident necessary
maintenance of interferon, and even ribavirin, in future
therapeutic regimens.
New Therapies for Hepatitis C
The current standard of care for patients with chronic
hepatitis C is the combination of pegylated interferon (IFN)
alpha and ribavirin. This treatment is effective in
approximately 80% of patients with hepatitis C virus (HCV)
genotype 2 or 3 infection, but less than 50% of those with
HCV genotype 1 [14]. Many new HCV drugs are at the
preclinical developmental stage and several are in clinical
development. Novel HCV therapies currently in development
schematically belong to four categories: novel IFNs,
alternatives to ribavirin, immune therapies, and specific and
nonspecific inhibitors of the HCV lifecycle.
Novel Therapies for Hepatitis C
Novel IFNs
New IFN alpha molecules are currently being developed.
They are expected to yield more potent antiviral effects, and
eventually more potent immunomodulatory effects, with
improved pharmacokinetic and pharmacodynamic properties
and, if possible, better tolerance. The potential interest in nonalpha IFNs is also being studied.
Albumin-linked IFN alpha
Albumin-linked IFN alpha (Albuferon®, Human Genome
Sciences and Novartis) is an IFN alpha-2b molecule attached
to a human albumin moiety which has a prolonged half-life
that allows dosing at intervals of 2 to 4 weeks. Albumin-linked
IFN alpha is able to induce an antiviral response in previous
nonresponders to pegylated IFN and ribavirin combination.
Results presented at the 42nd Annual Meeting of the European
Association for the Study of the Liver (EASL) have shown no
significant difference in the rates of sustained virological
response between patients receiving pegylated IFN alpha-2a
and ribavirin or various doses of albumin-linked IFN alpha
administered every two or four weeks with ribavirin.
Consensus IFN alpha
IFN alphacon-1 (Infergen, Amgen and InterMuneYamanouchi) is a synthetic recombinant “consensus” IFN
(cIFN) created by scanning the sequences of several natural
alpha IFNs and assigning the most frequently observed amino
acid in each corresponding position to the recombinant
molecule [15]. There is no clear evidence that cIFN is superior
to other alpha IFNs when both are given in equivalent doses
in terms of sustained virological response.
Other alpha IFNs in development
Multiferon (Viragen) is a highly purified, multi-subtype
natural human IFN alpha derived from human leukocytes
which has already been approved for use in HCV therapy in
several countries. Medusa® (Flamel Technologies) is a selfassembled poly-aminoacid nanoparticles system that can be
used as a protein carrier for the development of novel longacting native protein drugs. Medusa IFN alpha-2a and Medusa
IFN beta are currently in early clinical development. Various
types of orally administered IFNs alpha are also currently
being developed. Whether similar efficacy as with
subcutaneous administration can be achieved is under study.
Non-type I IFNs
IFN omega, like IFN alpha or beta, is a type 1 IFN. It shares
70% homology with IFN alpha and binds to the same receptor.
A stable, glycosylated form of IFN omega has been developed
(Intarcia) and has been administered to patients with chronic
hepatitis C in phase I and II trials. The results of a phase II trial
presented at the 42nd EASL Annual Meeting have shown
sustained virological response rates (HCV RNA below 50
international units (IU)/mL at week 12 post-therapy) of 36% in
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83
Table 1. Developing drugs that have a direct effect on HCV (adapted from [7,10])
Site of action
Compound
Input inhibitors
Anti HCV Immunoglobulin
(HCIg)
Monoclonal antibodies
(HCV-AB 68, HCVAB 65)
HCV-RNA
Antisense oligonucleotides
transcription
ISIS 14803
inhibitors
AVI-4065
RNA that catalyzes the
cleavage of the target RNA
(Ribozymes)
IRES small-molecule
inhibitors
Small Interfering RNA
(siRNA) or short hairpin
RNA (shRNA)
Post-transcription Serum proteinase inhibitors
processing
to NS3-4A
inhibitors
Commercial
name
Development
phase
Posology
Comments
Civacir
Phase II
200-400 mg/kg
Poor preliminary results
—
Phase I
—
—
Phase II
Transitory reduction of
viremia
Interrupted
—
Heptazyme
Phase I
Phase I
—
Current
Interrupted
VGX-410C
Phase I
—
Current
TT033
Pre-clinical
—
Parenteral use
—
Interrupted
BILN 2061
—
ACH-806/Gs-9132
VX-950
Phase II
(telaprevir)
VO 450-750 mg
PROVE –Telaprevir +
every 8 h
RBV+PEG-IFN: 88%
with RNA < 10 IU in 12
without vs. 52% PEG+RBV
HCV replication
inhibitors
Polymerase inhibitors NS5B
(RdRp)
Cyclophilin B inhibitors
NS5A inhibitors
Helicase inhibitors
Viral assembly
and release
Glucosidase inhibitor
(Iminosugar)
SCH 503034
(boceprevir)
ACH-806
and GS-9132
ITMN 191
ACH 1095
Nucleoside
analogs/
Nucleotides
Phase II
—
Phase I
To initiate
NM283
(Valopicitabine)
–An.Nucleos.
Phase II
R1626 –
An.Nucl.
Phase II
VO 200-400 mg
every 8 h
—
Resistance
Rash
SCH+PEG-IFN-α-2b +
RBV current
Interrupted
—
—
VO
—
—
Potent, but GI effects
VO
1.5-4.5g
BID
Marked reduction
PEG-IFN-RBV
combination under study
Anemia
MK-0608
Pre-clinical
Non-nucleoside HCV-796
—
inhibitors
Phase II with PEG-IFN
XTL 2125 Phase I
R7128 Phase I
DEBIO-025
Phase I
—
NIM 811
Phase I
A 689
Pre-clinical
—
A-831
Pre-clinical
—
QU633
—
—
recombinant human antibodies
UT-231B
Phase II future —
MX-3253
Phase II
—
(celgosivir)
www.bjid.com.br
Marked reduction
without resistance
Reduction of viremia
—
—
—
Poor results
PEG-IFN/RBV
combination
84
the IFN omega-ribavirin combination arm versus 6% in patients
treated with IFN omega alone. The manufacturer plans to
develop an implantable infusion pump that will release a steady
amount of IFN omega for approximately four weeks.
In spite of in vitro results that would support a potentiating
effect of IFN gamma on IFN alpha [16], the proof of concept
that IFN gamma could have a utility in the treatment of chronic
hepatitis C still remains to be made.
IFN lambda-1 (interleukin 29 or IL-29) is a member of a
novel family of cytokines that are distantly related to the IL10 family and type 1 IFNs. IFN lambda-1 exhibits dose- and
time-dependent inhibition of HCV replication in various
models, independent of types 1 and 2 IFN receptors and
induced pathways [17]. A pegylated form of IFN lambda will
soon enter clinical evaluation.
Alternatives to Ribavirin
Alternatives to ribavirin are needed that would have the same
effects as ribavirin on infection in combination with pegylated
IFN or other IFN molecules without its hemolytic properties.
However, the challenge for discovering such molecules is
hampered by the fact the antiviral mechanisms of ribavirin remain
poorly understood. Taribavirin (Valeant Pharmaceuticals) is an
amidine prodrug of ribavirin converted into ribavirin by adenosine
deaminases, which are primarily present in hepatocytes.
Taribavirin is thus preferentially taken up in the liver where it
serves for ribavirin delivery to the major site of HCV replication,
whereas it is not transported efficiently into red blood cells [18].
In two recent phase III trials in combination with pegylated IFN
alpha 2a and 2b, respectively, taribavirin at a flat dose of 600 mg
twice a day failed to achieve similar efficacy as weight-based
dosed ribavirin in patients with chronic hepatitis C of various
genotypes. The incidence of hemolytic anemia was however
significantly lower with taribavirin. New trials with higher doses
of taribavirin have been planned.
Immune Therapies
Various nonspecific immunomodulatory agents, including
thymosin alpha-1 (Thymalphasin, SciClone), IL-10 or histamine
(Maxym Pharmaceuticals) have been administered to patients
with chronic hepatitis C, with little success.
Vaccines can induce CD4+ and CD8+ T-cell responses to
HCV. Preclinical and early human studies indicate that
therapeutic vaccines using different forms of recombinant HCV
proteins together with various adjuvants could upregulate
both cellular and humoral immune responses in patients with
chronic hepatitis C [19]. However, there is currently no
evidence that therapeutic vaccines alone can result in changes
in HCV RNA levels. It remains to be determined whether
therapeutic vaccines could be useful in combination with
potent antiviral molecules.
Inhibitors of the HCV Life Cycle
Every step of the HCV lifecycle constitutes a potential target
for specific or nonspecific antiviral molecules. Many drugs are
at the preclinical developmental stage and several are in clinical
development, but initial trials using some of these inhibitors
alone have raised concerns about their tolerability and the
development of viral resistance. A number of specifically targeted
therapies are now also being tested in combination with
pegylated IFN alpha with or without ribavirin.
Inhibitors of the early steps of the HCV life cycle
Inhibition of HCV entry can be based on the use of specific
antibodies that neutralize infectious particles and prevent their
attachment to the receptor molecules. These include polyclonal
hepatitis C immune globulins, that have been administered to
prevent HCV infection in HCV-infected liver transplant recipients
with little success [20], and anti-HCV monoclonal antibodies
with high-affinity HCV neutralizing properties that reduce viral
replication by 0.3 to 1.0 log [21,22]. Small molecule entry
inhibitors can theoretically belong to two groups of drugs:
molecules that specifically fix onto the HCV surface structures
and neutralize the virus, or molecules that compete with
infectious viral particles at the receptor level. Unfortunately,
our understanding of HCV entry mechanisms remains
rudimentary, hampering the development of such molecules
inhibitors. Fusion could also become an interesting target for
novel therapies when its mechanisms are better understood.
HCV RNA translation inhibitors
Several nucleic acid-based strategies have been tested,
including
antisense
oligodeoxynucleotides,
phosphorodiamidate morpholino oligomers (PMO), or
ribozymes. All of them have been shown to potently inhibit
HCV translation in vitro, but have been disappointing in vivo
[23,24]. RNA interference initiated by small interfering RNAs
(siRNA) or short hairpin RNAs (shRNA) is very specific and
offers a potential to be used as antiviral against HCV. However,
because of their size and chemical composition, siRNAs and
shRNAs currently are not orally bioavailable and require
parenteral administration. Alternative strategies currently
target the three-dimensional functional internal ribosome entry
site (IRES) complexed with ribosomal subunits and viral and
cellular proteins with small molecule inhibitors.
Inhibitors of HCV post-translational processing
Highly selective, potent peptidomimetic inhibitors of HCV
NS3/4A proteinase have been designed. VX-950 or telaprevir
(Vertex Pharmaceuticals) and SCH 503034 or boceprevir
(Schering-Plough Corporation) have now advanced to phase
II clinical trials. Patients who received 750 mg of telaprevir
alone every 8 hours experienced a median 4.4-log reduction in
HCV RNA levels [25]. The drug is well tolerated over shortterm administration. However, viral breakthroughs occur
during the second week of telaprevir administration in patients
with low exposure to the drug. They are due to selection of
telaprevir-resistant variants. Combination with pegylated IFN
alpha with or without ribavirin could theoretically at least partly
prevent telaprevir resistance. This hypothesis is currently
www.bjid.com.br
under study in two phase II trials. Boceprevir appears to have
less potent antiviral properties than telaprevir in vivo at the
doses used to date [26]. In combination, the antiviral effect of
boceprevir appears to be additive to that of pegylated IFN
alpha-2b [26]. A phase II clinical trial is ongoing in combination
with pegylated IFN alpha and ribavirin, where high doses of
boceprevir are administered to treatment-naïve patients.
Other approaches have been developed to inhibit the NS3/
4A serine proteinase function. ACH-806/GS-9132 (Achillion
Pharmaceuticals and Gilead Sciences) inhibits binding of
NS4A to the NS3 proteinase, therefore inhibiting polyprotein
processing by preventing the formation of the active
proteinase complex. ACH-806/GS-9132 has potent in vitro
activity against HCV genotype 1 and administration of 300
mg twice daily for 5 days resulted in an average change in
HCV RNA level from baseline of -0.9 log [27]. The development
of this drug has been halted because of nephrotoxicity [27].
Inhibitors of HCV replication
Inhibitors of the RNA-dependent RNA polymerase (RdRp)
belong to two categories: nucleoside/nucleotide inhibitors,
that target the catalytic site of the enzyme, and non-nucleoside
inhibitors that target allosteric sites of the RdRp. Three RdRp
inhibitors have been administered to patients in clinical trials,
including two nucleoside and one non-nucleoside inhibitor.
Two have been withdrawn due to toxicity. R1626 (Roche
Products) induces a dose-dependent HCV RNA level reduction
[28]. At very high doses, HCV RNA level decrease reaches
more than 3 logs, but side-effects are frequent. R1626 in
combination with pegylated IFN alpha and ribavirin has
recently progressed into phase II of clinical development. Other
drugs have entered phase I clinical development.
The HCV RdRp has been reported to bind cyclophilin B, a
cellular peptidyl-prolyl cis-trans isomerase that apparently
regulates HCV replication through modulation of the RNA
binding capacity of RdRp. Synthetic, non-immunosuppressive
cyclophylin B inhibitors have been developed and are being
tested in patients with chronic HCV infection [29]. Side-effect
were however frequent and led to treatment withdrawal in
several cases [16]. Another cyclophilin B inhibitor, NIM 811
(Novartis) is currently being tested in a phase I trial [30,31].
Inhibitors of virus assembly and release
Iminosugars have been suggested to be able to cross
cellular membranes and concentrate in the endoplasmic
reticulum where they could competitively inhibit envelope
proteins glycosylation and interfere with viral assembly [32].
MX-3253 or celgosivir (Migenix) has a modest antiviral effect
on HCV in monotherapy. It is currently administered in
combination with pegylated IFN alpha and ribavirin in a phase
II clinical trial.
Questions and Issues with New Therapies
Among the many new avenues being explored, orally
administered antiviral drugs that specifically inhibit a step of
85
the HCV lifecycle have come under the spotlight. However,
although encouraging results have been published, serious
issues have been raised as to the antiviral potency of these
drugs, their tolerability, and the crucial problem of viral
resistance.
Antiviral Potency
Antivirals usually enter development as soon as they show
some degree of antiviral efficacy in one of the existing in vitro
models. However, in vitro antiviral activity does not always
translate into antiviral efficacy in vivo. There are several
examples of drugs that were highly potent in vitro but failed
when administered to patients. Unfortunately, studies with
“negative” results are rarely published, even though they
could teach us a lot. There are several possible reasons for
these in vitro/in vivo discrepancies, including the use of
poorly relevant preclinical models, misinterpretation of
preclinical data, poor pharmacokinetics, poor delivery of a
potentially potent drug to its target site, or the fact that the
target is not physically accessible in infected cells in vivo.
Tolerability
Small-molecule viral inhibitors have been under close
scrutiny for potential toxicity. The clinical development of
BILN 2061 (Boehringer-Ingelheim, Ingelheim, Germany), a
potent HCV NS3 serine protease inhibitor and the first drug of
this type to be administered to infected patients, has been
suspended because of myocardial toxicity in animals [33,34].
The development of an enormous number of potentially active
HCV drug candidates was stopped before they were given to
patients, owing to concerns raised by preclinical toxicity
studies in vitro and in animal models. The clinical development
of several drugs has been stopped because of their side-effect
profile, including valopicitabine (digestive side-effects), HCV796 (ALT elevations) and ACH-806 (nephrotoxicity). Other
drugs are still being developed in spite of serious side-effects.
R1626 has been shown to induce a dose-dependent reduction
of blood cell counts and hemoglobin levels after two weeks of
administration at high doses [28], and the outcome of these
effects is unknown if therapy is prolonged or if ribavirin is
used in combination. Apparent safety during short-term
administration does not guarantee that no serious adverse
effects will occur when the drug is given for several weeks or
months. Reesink et al. observed no serious adverse events
during 14 days of telaprevir administration [25]. However, a
recent commercial press release from Vertex Pharmaceuticals
(December 13, 2006) states that, in the PROVE 1 study, a phase
2b twelve-week clinical trial of the triple combination of
pegylated IFN alpha, ribavirin and telaprevir in treatment-naive
patients, 3% of the patients discontinued telaprevir because
of rash (rash was the most common reason for treatment
discontinuation). Drug-drug interactions may also be a
problem when HCV inhibitors are used in combination with
pegylated IFN, with or without ribavirin. In addition, although
synergy or additive efficacy may be expected, antagonism
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86
can also occur. Here again, in vitro studies may give clues as
to likely adverse effects but they cannot replace in vivo
studies.
Resistance
A surprising finding has been the frequency and early
timing of the emergence of resistance with all classes of antiHCV drugs. These results suggest that resistant variants are
preexisting, fit, and ready to be selected by any specific HCV
inhibitor. These findings disqualify HCV inhibitor
monotherapy and raise major ethical issues as to whether
naive or nonresponder patients should now be included in
trials of these drugs in monotherapy, as there is a risk that
they will be disqualified from future trials and therapies with
drug combinations.
Conclusion
At this point, it is clear that specific HCV inhibitors should
not be used alone. Combination therapy with oral antiviral
drugs will require company portfolios to contain more than
one such drug. And appropriate preclinical drug-drug
interaction studies will have to be performed before clinical
trials are initiated. This may take several years. In the
meantime, new strategies are needed to improve the results
of current HCV therapy. The “conservative“ approach aimed
at optimizing pegylated interferon-ribavirin therapy should
not be neglected. Preliminary results have shown that
increasing the dose and/or the number of injections of
pegylated IFN increases the response rate, and this is being
further explored in ongoing trials. Likewise, increasing the
dose of ribavirin has been shown to significantly improve
the cure rate, and the adverse effects of ribavirin can now be
partly controlled by using erythropoietin. Other trials are
addressing the best way of tailoring the duration of treatment
to the early virologic response (i.e. the HCV RNA decline at
week 4 or even week 2). Another option is to enhance
pegylated IFN-ribavirin efficacy by adding antiviral drugs
with an additive or synergistic antiviral effect. Such
combinations have the advantage of theoretically preventing
the onset of resistance to the inhibitor, through the antiviral
effect of IFN alpha. This may indeed happen in good IFN
responders, although it is unclear whether IFN inhibition
will be sufficient to avoid the emergence of resistance. In
addition, patients with little or no response to IFN will in
effect be receiving inhibitor monotherapy. Preliminary data
are encouraging and ongoing trials will show how well double
and triple combinations are tolerated, and whether the
responses persist.
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34. Reiser M., Hinrichsen H., Benhamou Y., et al. Antiviral efficacy of
NS3-serine protease inhibitor BILN-2061 in patients with chronic
genotype 2 and 3 hepatitis C. Hepatology 2005;41:832-5.
www.bjid.com.br
88
INSTRUCTIONS FOR AUTHORS
Manuscripts for submission to The Brazilian
Journal of Infectious Diseases should be sent to
Anastácio Queiroz de Sousa, Editor-in-Chief, Rua
Alfredo Magalhães, 04/Barra, 40140-140, SalvadorBahia-Brazil. Each manuscript will be assigned a
registration number, and the author notified that the
manuscript is complete and appropriate to begin the
review process.
Authors must indicate in a cover letter the address,
telephone number, fax number, and e-mail of the
corresponding author. The corresponding author will
be asked to make a statement confirming that the
content of the manuscript represents the views of the
co-authors, that neither the corresponding author nor
the co-authors have submitted duplicate or
overlapping manuscripts elsewhere, and that the items
indicated as personal communications in the text are
supported by the referenced person.
Each manuscript is to be submitted as an original
with three copies and four sets of illustrations. For
submission to Letters to the Editor or the Editor's
Corner, an original and one copy are required. For
submission to Clinical Infectious Diseases Images,
which are not intended as a vehicle for case reports,
all text should be in one double-spaced electronic
document, title (no more than eight words in the
title; no more than five authors may be listed,
inclusing name, highest academic degree, address,
e-mail address, telephone and fax number of each
author; the text should contain no more than 300
words. The other manuscripts are to be typed
double-spaced, including text, tables, references
and legends. All pages are to be numbered, with
the order of presentation as follows: title page,
abstract, text, acknowledgements, references,
tables, figure legends and figures. A running title of
not more than 40 characters should be at the top of
each page. The abstract should not exceed 250
words. References should be listed consecutively in
the text and recorded as follows in the reference list.
Authors (when there are fewer than five, record all
authors; when there are five or more, record the first
three, followed by et al.), title, journal, year, volume,
pages (first and last).
References must follow the format of the National
Library of Medicine as in Index Medicus and "Uniform
Requirements". Titles of journals not listed in Index
Medicus should be spelled out in full. Several examples
of specific references follow:
Article
(If the journal is not listed in Index Medicus)
Smith J.C., Charles R.S. Microbes and water filters.
Journal of Water Purification 1996;20:165-170.
(If the journal is in Index Medicus)
Watson B.M., Gupta R., Randal T., Starr S.
Persistence of cell-mediated immune responses
post-VZV immunization. J Infect Dis
1994;169:197-9.
Book Chapter
Taylor D.M., Personnet J. Epidemiology and natural
history of Helicobacter pylori infection. In: Blaser
M.J., Smith P.D., Ravdin J. eds. Infections of the
gastrointestinal tract. New York: Raven Press,
1994.
Book
Polak J.M., Van Noordan S. An introduction to
immunochemistry: current techniques and problems.
Oxford, UK: Oxford University Press, 1987.
Abstract
Blatt S.P., Butzin C.A., Lucey D.R., Melcher G.P.,
Hendrix C.R. Anergy status and CD4 CD29
memory T-cells predict progression to AIDS
(abstract PoB 3480). In: Program and abstracts:
VIII International Conference on AIDS
(Amsterdam) Amsterdam: CONGREX Holland,
1992.
Figures may be submitted as glossy photographs
or as high-quality laser prints on bond paper.
Manuscript style should follow accepted standards.
Please refer to the BJID for guidance. The final style will
be determined by the Medical Editor after it is reviewed
and accepted by the manuscript's corresponding author.
www.bjid.com.br
89
Evaluation of Manuscripts
STATEMENT OF
EDITORIAL POLICY
(BJID) is organized, guided and sponsored by the
Brazilian Society of Infectious Diseases.
Communications published in the BJID are to be
relevant in the broadest sense to all aspects of
microbiology, infection and immune response to
infectious agents.
The Editor will obtain reviews of each submitted
manuscript by two expert consultants, usually from
the Editorial Board of the BJID. Exceptions will be
made to this policy by the Editor only in cases in
which the content of the manuscript is not consistent
with the editorial policy of the Journal. Authors may
suggest appropriate consultants for review of the
manuscript. The review process will ordinarily
require two months.
Manuscripts may be submitted within designated
categories of communication including:
• original basic or clinical investigation;
• case presentation and discussion;
• brief reports of new methods or observations;
• clinical infectious diseases images;
• state-of-the-art presentations or reviews;
• letters to the editor concerning previous publications;
• editor's corner containing ideas, hypotheses and
comments.
Supplements to the BJID include articles under a
unifying theme, such as those summarizing presentations
of symposia or focusing on a specific pathogenic
process or antimicrobial agent. These will be added to
the regular bi-monthly publication as appropriate and
will be peer reviewed in the same manner as submitted
manuscripts.
Publication
The BJID is published bi-monthly (February,
April, June, August, October, December) by
Contexto Publishing Inc., Rua Alfredo Magalhães,
04/Barra, 40140-140, Salvador-Bahia-Brazil. The
editors of the Journal reserve the right to edit
manuscripts for clarity, grammar and style. Authors
will have an opportunity to review these changes
prior to creation of galley proofs. Changes in content
after galley proofs are sent for review will require
charges to the author. The BJID does not accept
articles which duplicate or overlap publications
elsewhere.
www.bjid.com.br
90
Checklist for Submitted Manuscripts
1. Please provide a cover letter with your submission, specifying the corresponding author as well as an
address, telephone and facsimile number.
2. Submit four copies of your manuscript (original plus three copies and a disk of the manuscript - Word
Perfect/Word for Windows), each with a complete set of original illustrations.
3. The entire manuscript (including tables and references) must be typed double-spaced and printed on standardsized paper. The left and right margins must be at least 3 cm.
4. The entire manuscript must be typed in a font size of at least 12 points.
5. Please number pages beginning with the title page (title page is page 1).
6. The order of appearance of material in all manuscripts should be as follows: title page, abstract, text,
acknowledgements, references, tables, legends for figures, figures.
7. The title page must include a title of not more than three printed lines (250 letters and spaces), authors (no
titles or degrees), institutional affiliations, a running headline of not more than 40 letters and spaces, a name and
complete address to which correspondence and reprint requests should be sent, and footnotes indicating sources
of financial support and changes of address.
8. Abstract (maximum 250 words) must be on a separate page before the introduction. Do not submit an
abstract with correspondence.
9. Acknowledgements of persons who assisted the authors should be included on the page preceding the
references.
10. References must begin on a separate page.
11. References must be cited on (not above) the line of text and in brackets instead of parentheses, e.g., [7,8].
12. References must be numbered in the order in which they appear in the text. References not cited in the text
cannot appear in the reference section. References that are only or are first cited in a table or figure are numbered
according to where the table or figure is cited in the text. For instance, if a table is placed after reference 8, a new
reference cited in table 1 would be reference 9.
13. Reference citations must follow the format established by the "Uniform Requirements for Manuscripts
Submitted to Biomedical Journals" (see examples in Instructions to Authors).
14. If you reference your own unpublished work (i.e., an "in press" article) in the manuscript that you are
submitting, you must enclose three copies of the "in press" article and an acceptance letter from the journal.
15. If you cite unpublished data that are not your own, you must provide a letter of permission from the author
of that publication.
16. Please provide four glossy or laser-produced prints of each figure that you are submitting. Label all figures
clearly with first author's name and figure number (place typed label on the back of the figure).
17. Provide a figure legend for each figure. Figure legends must be on a separate page at the end of the
manuscript.
Please refer to Instructions to Authors for further information regarding the section to be submitted
the manuscript, title page, abstract, references, tables and figures, and style.
www.bjid.com.br

the brazilian journal of infectious diseases

Transcription

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