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ISSN 1413-8670

Volume 12

•

Number 5

•

October 2008

THE BRAZILIAN JOURNAL OF INFECTIOUS DISEASES An Official Publication of the Brazilian Society of Infectious Diseases

EDITOR Anastácio Q. Sousa

PUBLISHED BY CONTEXTO

October 2008 Printed in Brazil www.bjid.com.br

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BJID 2008; 12 (October)

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 2008-2009

President Juvêncio José Duailibe Furtado Vice President Marcelo Simão Ferreira First Secretary Mauro José Costa Salles Second Secretary Beatriz Gilda Jegerborn Grinsztejn First Treasurer Francisco Bonasser Filho Second Treasurer Carlos Roberto Brites Alves Scientific Coordinator Dr. João Silva de Mendonça Computing Coordinator Hamilton Antonio Bonilha de Moraes Coordinator’s Disclosure Policy Dr. Thais Guimarães

Federal Alagoas - Rosane Maria S. Brandão Amazonas - Eucides Batista da Silva Bahia - Adriano Oliveira Ceará - Roberto da Justa Pires Neto Distrito Federal - Julival Ribeiro Espírito Santo - Nilo F. Rezende Vieira 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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BJID 2008; 12 (October)

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)

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The Brazilian Journal of Infectious Diseases is an official publication of the Brazilian Society of Infectious Diseases and is published bi-monthly by Contexto - Rua Alfredo Magalhães, 04/Barra, 40140-140, Salvador-BahiaBrazil. The editorial offices are at Contexto.

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Permissions Copyright 2008 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved. Except as authorized in the accompanying statement, no part of the BJID may be reproduced in any form or by any electronic or mechanical means, including information storage and retrieval systems, without the publisher's written permission. Authorization to photocopy items for internal or personal use, or the internal or personal use by specific clients is granted by The Brazilian Journal of Infectious Diseases and Contexto Publishing for libraries and other users. This authorization does not extend to other kinds of copying such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. COVER: Figure: Left: HE staining shows a chronic granulomatous (CG) dermatitis with fibrinoid necrosis and a thickened nerve. Faraco and Grocott negative. Right: HE: Chronic granulomatosus reaction with extensive caseous necrosis and neuritis on dermis and hypodermis. Article: Leprosy with Necrosis in Granulomatous Reaction by Maria Angela B. Trindade, et al. Braz J Infect Dis 2008;12(5):460.

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THE

BR AZILIAN BRAZILIAN

JOURNAL

OF

INFECTIOUS

Volume 12 • Number 5

DISEASES October 2008

Brief Communications

Molecular Investigation of a Fungemia Outbreak Due to Candida parapsilosis in an Intensive Care Unit ............. 395

Quinine Levels in Patients with Uncomplicated Falciparum Malaria in the Amazon Region of Brazil ........................... 353

Murat Dizbay, Ayse Kalkanci, Busra Ergut Sezer, Firdevs Aktas, Sibel Aydogan, Isil Fidan, Semra Kustimur and Takashi Sugita

José Luiz Fernandes Vieira, Larissa Maria Guimarães Borges, Margareth Tavares Silva Nascimento and Andreza L.S. Gomes

Lamivudine for Chronic Hepatitis B: A Brief Review ...... 355 Emilio Palumbo

Hepatitis C Virus Detection in the Semen of Infected Patients ......................................................................... 358 Norma de Paula Cavalheiro, Ana Carolina de Oliveira Santos, Carlos Eduardo Melo, Suzana Rie Morimitsu and Antonio Alci Barone

Treatment of Invasive Fungal Infections: Stability of Voriconazole Infusion Solutions in PVC Bags ................ 400 Andréa I.H. Adams, Lucia N. Morimoto, Leonardo Z. Meneghini and Ana M. Bergold

Rates of Antimicrobial Resistance in Latin America (2004-2007) and in vitro Activity of the Glycylcycline Tigecycline and of Other Antibiotics ........................................................... 405 Flávia Rossi, Patricia García, Bernardo Ronzon, Daniel Curcio and Michael J. Dowzicky

Inadequate Timing Between Corticosteroid and Antibiotics Applications Increases Mortality Due to Sepsis ........... 416

Original Papers Histological Response Study of Chronic Viral Hepatitis C Patients Treated With Interferon Alone or Combined With Ribavirin ......................................................................... 362 Kleber Prado, Rosely Patzina, Denise Bergamaschi and Roberto Focaccia

Cost-Effectiveness of Entecavir versus Lamivudine for the Suppression of Viral Replication in Chronic Hepatitis B Patients in Brazil ............................................................ 368

Marcus Vinícius Telles Fadel, João Carlos Repka, Cláudio Leinig Pereira da Cunha and Maria Terezinha C. Leão

Comparison of PCR-Based Molecular Markers for the Characterization of Proteus mirabilis Clinical Isolates .... 423 Lessandra Michelim, Gabriela Muller, Jucimar Zacaria, Ana Paula Longaray Delamare, Sérgio Olavo Pinto da Costa and Sergio Echeverrigaray

Anna Maria N. Costa, Gilbert L.’Italien, Marcelo Eidi Nita and Evaldo Stanislau A. Araujo

Ribotyping, Biotyping and Capsular Typing of Haemophilus influenzae Strains Isolated from Patients in Campinas, Southeast Brazil ............................................................. 430

Genetic Characterization and Evolutionary Inference of TNFα Through Computational Analysis .............................. 374

Marcelo Lancellotti, Fernanda de Pace, Eliana Guedes Stehling, Maria Cecília Barisson Villares, Marcelo Brocchi and Wanderley Dias da Silveira

Gauri Awasthi, Suchita Singh, A.P. Dash and Aparup Das

Prevalence of Resistance-Associated Mutations in Human Immunodeficiency Virus Type 1-Positive Individuals Failing HAART in Rio de Janeiro, Brazil .................... 380 Rafael Brandão Varella, Selma Baía Ferreira, Márcia Braga de Castro, Marisa Dias Tavares and Mariano Gustavo Zalis

Invasive Aspergillosis in Hematopoietic Stem Cell Transplant Recipients: A Retrospective Analysis ........................... 385 Viviane Maria Hessel Carvalho-Dias, Caroline Bonamin Santos Sola, Clóvis Arns da Cunha, Sílvia Emiko Shimakura, Ricardo Pasquini and Flávio de Queiroz-Telles

An Outbreak of Candida spp. Bloodstream Infection in a Tertiary Care Center in Bogotá, Colombia ...................... 390 Carlos A. DiazGranados, Adriana Martinez, Ceneth Deaza and Sandra Valderrama

Postnatal Acquired Toxoplasmosis Patients in an Infectious Diseases Reference Center ............................................ 438 Cassius Schnell Palhano Silva, Elizabeth de Souza Neves, Eliezer Israel Benchimol and Danielle Ribeiro de Moraes

Case Reports Tatumella ptyseos Causing Severe Human Infection: Report of the First Two Brazilian Cases ..................................... 442 Paulo Sérgio Gonçalves da Costa, Juliana Monteiro de Castro Mendes and Geyza Machado Ribeiro

Ventilator-Associated Pneumonia with Col–S Strains: A Successful Comeback of Colistin! .................................. 444 Mukhyopadhyay C., Krishna S., Vandana K.E., Shenoy A. and Bairy I.

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Extensively Drug-Resistant Tuberculosis: A Case Report and Literature Review ........................................................... 447

Images in Clinical Infectious Diseases

João Alves de Araújo-Filho, Arioldo Carvalho Vasconcelos-Jr, Eduardo Martins de Sousa, Colombina da Silveira, Elisangela Ribeiro, André Kipnis and Ana Paula Junqueira-Kipnis

Leprosy with Necrosis in Granulomatous Reaction ....... 460

Primary Gastric Fundus Tuberculosis in Immunocompetent Patient: A Case Report and Literature Review ................... 453

Instructions for Authors

Fahmi Yousef Khan, Ahmed AlAni, Ammar Al-Rikabi, A. Mizrakhshi and Mohamed El-Mudathir Osman

Maria Ângela Bianconcini Trindade, Brandt H., Teixeira R., Sotto M.N. and Fleury R.N.

Statement of Editorial Policy

Acute Hepatitis Due to Dengue Virus in a Chronic Hepatitis Patient ............................................................................ 456 Souza L.J., Coelho J.M.C.O., Silva E.J., Abukater M., Almeida F.C.R., Fonte A. S. and Souza L.A.

Checklist for Submitted Manuscripts

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Quinine Levels in Patients with Uncomplicated Falciparum Malaria in the Amazon Region of Brazil José Luiz Fernandes Vieira 1, Larissa Maria Guimarães Borges 2 , Margareth Tavares Silva Nascimento 2 and Andreza de L.S. Gomes3 1 Professor of Toxicology of the Department of Legal Medicine of Federal University of Pará; 2Graduate Student (Master’s Degree Program in Tropical Disease) of Federal University of Pará; 3Undergraduated Student of Federal University of Pará; Belém, PA, Brazil

We examined the plasmatic concentrations of quinine in patients with uncomplicated falciparum malaria in an endemic area of the Amazon region in Brazil in a prospective clinical trial, in which a standard three-day course of oral quinine plus doxycycline was used. We measured the quinine in the plasma samples on days 0 and 3by high performance liquid chromatography. The mean concentration of quinine was 6.04 ±2.21 μg/mL in male patients and 5.98 ±1.95 μg/mL in female patients. No significant differences in quinine concentration were observed between these two groups. All samples collected before starting treatment were negative for quinine. This information could help in the development of strategies for the rational use of antimalarial drugs in Brazil. Key-Words: Falciparum malaria, quinine, treatment.

The monitoring of quinine blood concentration is an important approach for better understanding the epidemiology of falciparum malaria, especially for a clear distinction between treatment failure, i.e. the absence of resolution of clinical signs after antimalarial treatment associated with inadequate drug concentration, and true resistance to an antimalarial drug [1,2]. Much work has been done to determine how blood quinine concentration profiles correlate with subsequent therapeutic responses in malaria patients; however, the precise therapeutic range of plasma quinine levels remains uncertain, because the pharmacokinetics and the therapeutic response to quinine varies with drug formulation, patient age and immunity, the sensibility of the malaria strain [3,4], the fraction bound to α1-acid-glycoprotein, parasite clearance in the course of the disease and interracial differences in metabolism [5]. However, some authors have stated that continuous concentrations ≥ 6 μg/mL are required to ensure cure [2,4]. There is a lack of knowledge about the plasmatic concentration of quinine in Brazilian falciparum-malaria patients that have been treated with the standard threeday course of oral quinine. We examined the plasmatic concentrations of quinine in patients with uncomplicated falciparum malaria in an endemic area of the Amazon region, in a prospective clinical treatment trial [6]. This study was conducted from January 2006 to January 2007, on 15 adult male and 15 adult female patients with uncomplicated P. falciparum malaria admitted to the public Hospital of Tucurui, Amazonas, Brazil. Patients with severe or mixed malaria and pregnancy were excluded. Patients who had taken any antimalarial drugs within the previous 48 hours were also excluded. All patients were monitored for at least 28 days within the malaria Received on 12 May 2008; revised 10 September 2008. Address for correspondence: Dr. Jose Luiz Fernandes Vieira. Toxicology Laboratory- Federal University of Pará. Rua Augusto Corrêa 01. Phone: 559132017733. E-mail: [email protected]. Belém, Para, Brazil. The Brazilian Journal of Infectious Diseases 2008;12(5):353-354. © 2008 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

transmission area. This study was approved by the ethics committee of the Tropical Medicine Center of Para Federal University. Informed consent was obtained from each subject. After clinical assessment and disease confirmation through microscopic examination of blood smears, all patients were treated with the first line standard threeday oral treatment regimen of quinine (750 mg), twice a day, followed by doxycycline hydrochloride (100 mg), twice a day, for five days and primaquine phosphate (45mg) on the last day [6]. Reappearance of infection was assessed in these patients for at least 28 days. Venous blood samples were taken for quinine level determination before and on the third day after treatment began. All blood samples were taken before quinine intake. The samples (3 mL) were collected in heparinized tubes and immediately centrifuged at 1500 x for 15 minutes. The plasma samples were stored at -20 0C until analysis. The Student’s t test was used to compare the two groups; P< 0.05 was considered significant. Quinine was analyzed by high performance liquid chromatography, as described previously [7]. The analytical procedure validated in our laboratory gave within-day and day-to-day coefficients of variation of 6.7 and 8.1%, respectively. Mean extraction recovery of the quinine was 95%. The stability of blank plasma spiked with quinine was 60 days under the conditions described above. Primaquine, doxycycline mefloquine, amodiaquine, acetaminophen and chloroquine do not interfere in the detection of quinine. All patients recovered following treatment. The mean time for parasite clearance was 70.4 hours, varying from 28 to 108 hours. None of the patients had recrudescent infection, checked until 28 days after treatment. The mean concentration of quinine in the male plasma samples in the steady state was 6.04 ± 2.21 μg/mL, ranging from 3.08 to 10.04 μg/mL; in the female samples it was 5.98 ± 1.95 μg/mL, ranging from 2.78 to 9.17 μg/mL. There were no significant differences between males and females in terms of quinine concentration in the plasma. These levels were similar to what was found in other studies, with plasmatic

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Quinine Levels and Uncomplicated Falciparum Malaria

quinine concentration during treatment of falciparum malaria varying from 5.0 to 10.0 μg/mL; they are also similar to steady state concentrations of quinine in African and Asian patients with uncomplicated falciparum malaria [2,8]. All samples collected before treatment were negative for quinine and confirmed drug absence. This fact is commonly observed in the Amazon region; it could favor the development of resistance of P. falciparum to drugs used in the treatment of this disease. Quinine concentrations must remain above levels that inhibit parasite multiplication throughout the course of treatment in order to eradicate infection, because there is a significantly higher probability that a resistant strain will emerge if the drug is present at suboptimal concentrations. It is very important to provide an adequate treatment course and to maintain complete adherence to the prescribed drug regimen in order to optimize cure rates and reduce the risks of side effects [1,3,5]. This information could be useful for developing strategies for the rational use of antimalarial drugs in Brazil.

BJID 2008; 12 (October)

References 1. 2.

3.

4.

5.

6. 7.

8.

WHO. World Health Organization- Guidelines for the treatment of malaria. Switzerland. 2006. Pukrittayakamee S., Wanwimolruk S., Stepniewska K., et al. Quinine Pharmacokinetic- Pharmacodynamic Relationships in uncomplicated falciparum malaria. Antimicrob Agents Chemother 2003;47:3458-63. Pukrittayakamee S., Looareesuwan S., Keeratithakul D., et al. A study of the factors affecting the metabolic clearance of quinine in malaria. Eur J Clin Pharmacol 1997;52:487-93. Supanaranond W., Davis T.M., Pukritayakamee S., et al. Disposition of oral quinine in acute falciparum malaria. Eur J Clin Pharmacol 1991;40:49-52. White N.J. Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrob Agents Chemother 1997;41:1413-22. Brasil. Fundação Nacional de Saúde. Manual de Terapêutica da Malária. 2nd. Brasilia, 2001. Dua K.V., Sarin R., Prakash A. Determination of quinine in serum, plasma, red blood cells and whole blood in healthy and Plasmodium falciparum malaria cases by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1993;614:87-93. Flanag K.L., Sharp M.B., Doherty T., Whitty C.J.M. Quinine levels revisited: the value of routine drug level monitoring for those on parenteral therapy. Acta Trop 2006;97:233-7.

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Lamivudine for Chronic Hepatitis B: A Brief Review Emilio Palumbo Pediatrics Department of Sondrio Hospital; Italy

Until recently, the only generally approved treatment for chronic hepatitis B was alpha-interferon; however, it gives only moderate efficacy in terms of sustained response (biochemical, virological and histological). In fact, only 20% to 40% of treated patients respond to therapy, with lower percentages (~ 10%) among patients infected with precoremutant strains of HBV (HBeAb HBV-DNA positive). The FDA of the USA approved the use of lamivudine in adult patients affected by chronic hepatitis B in 1998. In this review, we focused on the pharmacokinetic and pharmacodynamic properties and efficacy and tolerability of lamivudine in the treatment of chronic hepatitis B cases that are both HBeAg and anti-HBe-positive. Key-Words: Lamivudine, hepatitis, HBV.

Despite the use of HBV vaccine, chronic hepatitis B virus (HBV) infection occurs in approximately 5% of the global population. This infection, if persistent, may lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma in 25 to 40% of infected patients and is among the 10 main causes of death throughout the world [1,2]. Until recently, the only generally approved treatment for chronic hepatitis B was alpha-interferon; however, it has demonstrated only moderate efficacy in terms of sustained response (biochemical, virological and histological). In fact, only 20 to 40% of treated patients respond to therapy, with lower percentages (~10%) among patients infected with precore-mutant strains of HBV (HBeAb HBV-DNA positive) [3,4]. This form, prevalent in the Mediterranean region (~90% of all patients with HBV infection), is due to a mutation at nucleotide 1896 in the precore region of the HBV-DNA genome. The result of this mutation is a stop codon that blocks HBeAg synthesis but still permits HBV replication and hepatitis B core antigen production, leading to persistent viremia and persistent or intermittent elevated serum alanine aminotransferase (ALT) levels, with frequent evolution of the disease into cirrhosis and hepatocellular carcinoma. The suboptimal response of this form to alpha-interferon, with a high rate of non-responders or relapsers, has led to research and development of new antiviral drugs that could be used as alternative therapies. The use of nucleotide analogues is a milestone in the treatment of chronic hepatitis B (CHB). The FDA of the USA approved the use of lamivudine in adult patients in 1998. This drug is advantageous because of oral administration and good safety parameters; however, it induces a sustained response (after withdrawal of therapy) in only a minority of patients. Thus, the treatment should be given to most patients for long periods. In addition, the long-term efficacy of lamivudine is Received on 5 May 2008; revised 15 September 2008. Address for correspondence: Dr. Emilio Palumbo. Department of Pediatric, Hospital of Sondrio. Via Stelvio, 25. Zip code: 23100. Sondrio, Italy. E-mail: [email protected]. The Brazilian Journal of Infectious Diseases 2008;12(5):355-357. © 2008 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

limited by the frequent emergence of drug-resistant HBV mutants [5-7]. Adefovir is a nucleotide analogue associated with a low frequency of resistance; however, its antiviral efficacy is not optimal [8,9]. In this review, we focused on the pharmacokinetics and pharmacodynamics and efficacy and tolerability of lamivudine treatment of chronic hepatitis B cases that are both HBeAg and anti-HBe-positive. Relevant literature was identified through searches of MEDLINE (2002-October 2006). Search terms included, but were not limited to, lamivudine, hepatitis B, pharmacology, pharmacokinetics, adverse events, and therapeutic use. Pharmacokinetic and Pharmacodynamic Properties Lamivudine (3TC), the negative enantiomer of 2'-deoxy-3'thiacytidine, is a dideoxynucleoside analogue used in combination with other agents in the treatment of human immunodeficiency virus type 1 (HIV-1) infection and as monotherapy in the treatment of hepatitis B virus (HBV) infection. It is a nucleoside analog that inhibits HBV-DNA synthesis. Lamivudine undergoes anabolic phosphorylation by intracellular kinases to form lamivudine 5'-triphosphate, the active anabolite that prevents HIV-1 and HBV replication by competitively inhibiting viral reverse transcriptase and terminating proviral DNA chain extension. The pharmacokinetics of lamivudine are similar in patients with HIV-1 or HBV infection, and in healthy volunteers. The drug is rapidly absorbed after oral administration, with maximum serum concentrations usually attained 0.5 to 1.5 hours after administration. The absolute bioavailability is approximately 82 and 68% in adults and children, respectively. Lamivudine systemic exposure, as measured by the area under the serum drug concentration-time curve (AUC), is not altered when it is administered with food. Lamivudine is widely distributed in total body fluid, the mean apparent volume of distribution (Vd) being approximately 1.3 L/Kg following intravenous administration. In pregnant women, lamivudine concentrations in maternal serum, amniotic fluid, umbilical cord and neonatal serum are comparable, indicating that the drug diffuses freely across the placenta. In postpartum women, lamivudine is secreted into breast milk. The concentration of lamivudine in cerebrospinal fluid (CSF) is low to modest, reaching 4% to 8%

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Lamivudine for Chronic Hepatitis B

of serum concentrations in adults and 9% to 17% of serum concentrations in children measured at two to four hours after administration. In patients with normal renal function, about 5% of the original compound ismetabolized to the transsulphoxide metabolite, which is pharmacologically inactive. In patients with renal impairment, the amount of transsulphoxide metabolite recovered in the urine increases, presumably as a function of decreased lamivudine elimination. As approximately 70% of an oral dose is eliminated in the urine as unchanged drug, the dose needs to be reduced in patients with renal insufficiency. Hepatic impairment does not affect the pharmacokinetics of lamivudine. Systemic clearance following single intravenous doses averages 20 to 25 L/h (approximately 0.3 L/h/Kg). The elimination half-life of lamivudine is approximately five to seven hours, and the in vitro intracellular half-life of its active 5'-triphosphate anabolite is 10.5 to 15.5 hours and 17 to 19 hours in HIV-1 and HBV cell lines, respectively. Efficacy and Tolerance Randomized controlled trials have demonstrated the efficacy of lamivudine in the treatment of HBeAg-positive and HBeAg-negative CHB. One randomized, placebocontrolled trial showed that almost all patients treated with lamivudine (98%) had a reduction of serum HBV-DNA levels. Serum HBV-DNA levels were undetectable after lamivudine therapy in 44% of patients, compared with 16% of patients treated with a placebo. After one year of treatment, the HBeAg seroconversion rates were 17 and 6% in the lamivudine and placebo groups, respectively. ALT levels were normalized in 41% of the lamivudine-treated patients compared with 7% of the placebo group. Histological improvement was observed in 52% of the lamivudine group compared with 23% of the placebo group [5]. The rates of virologic, biochemical and histological response in another randomized controlled trial were similar, with an HBeAg anti-HBeAb seroconversion rate of 21% [6]. The tolerability and safety of lamivudine were excellent, and the frequency of adverse events was similar to that of the placebo group. Lamivudine therapy seems to be well-tolerated for up to five years; however, data on long-term therapy with lamivudine are limited to a small number of patients. The main limitation of lamivudine treatment is the high rate of viral resistance due to mutations in the YMDD motif of the HBV polymerase gene. Indeed, even if the HBeAg seroconversion rate is increased by continuing treatment, the frequency of resistance to lamivudine increases with time: 24% at one year, 38% at two years, 50% at three years, and 67% at four years [7]. The most important mutation is substitution of valine or isoleucine for methionine. In many patients, this mutation isaccompanied by a second mutation, in which methionine is substituted for leucine in an upstream region (rtL180M). Lamivudine resistance is more likely to occur in patients with high baseline levels of serum HBV-DNA. Emergence of lamivudine resistant mutants is usually associated with a breakthrough of hepatitis, resulting in

BJID 2008; 12 (October)

moderately increased levels of serum HBV-DNA and ALT, although these levels may remain lower than baseline (pretreatment) for several months. In another investigation, 76 HBeAg-negative patients with CHB were given 100 mg LAM daily in a five-year follow-up study [10]. The incidence of YMDD was 39% at month 12, 54% at month 24 and 57% at month 36; at the same time, the response declined from 67% at six months to 51%, 34% and 29% after 12, 24 and 36 months, respectively. A complete analysis at month 24 of follow-up was available. Increases in HBV-DNA and ALT levels were apparent in patients with YMDD variants. Among the patients who did not develop YMDD variants, 72% had normal ALT and negative HBVDNA, whereas only 5% of the patients who developed YMDD mutants maintained normal ALT levels after one year. In a recent study the efficacy of LAM administered for three years in patients with chronic active anti-HBe-positive hepatitis was evaluated. Thirty-four patients with chronic active anti-HBe-positive hepatitis were treated with LAM (100 mg) once daily for three years. Before treatment, all patients had serum ALT levels >two times normal levels for >six months and HBV DNA positivity >5 pg/mL, as determined by a sandwich hybridization test for nucleic acids. Both ALT and HBV DNA were monitored during therapy. After 12 months of therapy, 24 of 34 patients (70.6%) showed evidence of HBV DNA clearance and normal ALT levels; 22 of 34 (64.7%) and 19 of 34 (55.8%) patients maintained a complete response after two and three years of therapy, respectively. Long-term LAM therapy (>one year) was not associated with an increase in the response of initially nonresponding patients. The YMDD variant emerged in 17.6% of patients in the first year, in 35.2% during the second year, and in 52.9% during the third year of treatment. LAM was well tolerated during the three-year therapy in all patients. In patients with chronic active anti-HBe-positive hepatitis, the LAM response rate tended to decrease over time due to the emergence of YMDD variants [11]. Conclusions The goal of therapy for patients with HBV infection is to prevent the progression of liver disease to cirrhosis and hepatic cell cancer. In recent years, progress has been made in the treatment of CHB. Nucleos(t)ide analogs such as lamivudine have been approved as initial therapy for CHB. Currently, lamivudine is the first line of treatment of chronic hepatitis B; however, efficacy is limited by the high frequency of resistance. Recent preliminary results show that adefovir dipivoxil and entecavir could be potent analogs for the treatment of HBV. Further studies are being made to assess the long-term efficacy and safety of these drugs [12,13]. References 1. Chu C.M., Liau Y.F. Natural history of chronic B virus infection: an immunopathological study. J Gastroenterol Hepatol 1997;12, S218-S22.

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Lamivudine for Chronic Hepatitis B

2. Ganem D., Prince A.M. Hepatitis B virus infection-natural history and clinical consequences. N Engl J Med 2004;350:1118-29. 3. Olivero F., Santantonio T., Bellati G., et al. Long-term response to therapy of chronic anti-Hbe-positive hepatitis B is poor independent of type and schedule of interferon. Am J Gastroenterol 1999;94:1366-72. 4. van Zonneveld M., Honkoop P., Hansen B.E., et al. Long-term follow-up of alpha-interferon treatment of patients with chronic hepatitis B. Hepatology 2004;39:804-10. 5. Lai C.L., Chien R.N., Leung N.W., et al. A one year trial of lamivudine for chronic hepatitis B. N Engl J Med 1998;339:61-8. 6. Dienstag J.L., Schiff E.R., Wright T.L., et al. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med 1999;341:1256-63. 7. Lau D.T., Khokhar F., Doo E., et al. Long-term therapy of chronic hepatitis B with lamivudine. Hepatology 2000;32:828-34.

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8. Zeng M., Mao Y., Yao G., et al. A double-blind randomized trial of adefovir dipivoxil in Chinese subjects with HBeAg. positive chronic hepatitis B. Hepatology 2 0 0 6; 4 4 ( 1 ) : 1 0 8 - 1 6 . 9. Hadziyannis S.J., Tassopoulos N., Heathcote E.J., et al. Adefovir dipivoxil for the treatment of hepatitis B e antigen-negative chronic hepatitis B. N Engl J Med 2003;348:800-7. 10. Rizzetto M., Tassopoulos N.C., Goldin R.D., et al. Extended lamivudine treatment in patients with HBeAg-negative chronic hepatitis B. J Hepatol 2005;42(2):158-62. 11. Scotto G., Palumbo E., Fazio V., et al. Prolonged lamivudine treatment in patients with chronic active anti-HBe positive hepatitis. Am J Ther 2006;13(3):218-22. 12. Hadziyannis S.J. New developments in the treatment of chronic hepatitis B. Expert Opin Biol Ther 2006;6(9):913-21. 13. Zhou X.X., Littler E. Nucleoside analogs as anti-HBV agents. Curr Top Med Chem 2006;6(9):851-65.

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Hepatitis C Virus Detection in the Semen of Infected Patients Norma de Paula Cavalheiro, Ana Carolina de Oliveira Santos, Carlos Eduardo Melo, Suzana Rie Morimitsu and Antonio Alci Barone Laboratory of Hepatitis LIM-47 – Department of Infectious Diseases of Clinical Hospital of University of São Paulo; São Paulo, SP, Brazil

Though HCV infection is a serious public health problem, some aspects of its biology are still not well understood, such as its transmission through seminal fluid and sexual transmission. We looked for HCV in the semen of infected patients. Thirteen patients were included. Semen fractions (seminal plasma, leukocytes and spermatozoa) were separated with 45% and 90% Percoll gradients. The HCV-RNA in blood and semen fractions was extracted using the same protocol (AMPLICOR Roche) and was detected using the qualitative Roche Amplicor test and by agarose gel electrophoresis, with ethidium bromide staining. The mean age of the patients was 40.7 years. Risk factors for the acquisition of HCV included injectable and inhaled drug use in six (42.8%), blood transfusion in four (28.6%), and no risk factors in four (28.6%) patients. Genotype 1 was detected in 62% of the patients, followed by genotype 3 in 23% and genotype 2 in 15%. All blood samples were positive, regardless of the technique used for detection. All semen samples identified by Roche Amplicor and analyzed by agarose gel electrophoresis were negative. Among the 52 semen samples (total and fractions) identified by the Roche Amplicor method, 45 (87%) were inhibited. A negative result was recorded for one (1.9%) total semen sample, one (1.9%) leukocyte and four (7.7%) seminal plasma fractions. Only one (1.9%) sample of the spermatozoon fraction was positive. The results obtained suggested false-negative reactions for the semen samples. Key-Words: HCV, PCR, sexual transmission, semen.

Various studies suggest that the risk of sexual transmission of hepatitis C virus (HCV) is minimal or even inexistent, with its incidence ranging from 0 to 3% [1,2]. The first report discussing the sexual transmission of HCV, in which multiple sex partners were considered to be a risk factor, was published by Alter et al. in 1989 [3]. As is the case for sexual transmission, the risk of HCV transmission through seminal fluid is also controversial. Risk factors that may increase the probability of transmission are the type of relationship, since monogamic couples tend to present lower transmission rates than individuals reporting sex with multiple partners, sexual relations that involve trauma, co-infection with acquired immunodeficiency virus (HIV), partners using drugs, associated sexually-transmitted diseases, paid sex, and a long-term relationship (>10 years) with an HCV-positive partner [4-6]. The direction of sexual transmission of HCV from men to women has been reported by Rooney and Gilson [7], who showed that the estimated risk of HCV infection is 3.7 times higher in women with HCV-positive partners. Cavalheiro et al. [8] studied a series of 24 couples with a diagnosis of hepatitis C; there was an average viral similarity of 98.3% for 22/24 (91.7%) couples. In that study, the NS5b-HCV region was chosen for phylogenetic analysis. Nine couples attracted attention because the women did not report any risk factor for acquisition of the virus, whereas all nine men reported one or more risk factors. In this case, the average genomic similarity was 98%. That study supports the hypothesis of infection from men to women. Received on 26 May 2008; revised 10 August 2008. Address for correspondence: Dr. Norma de Paula Cavalheiro, Ph.D. Av. Dr. Enéas de Carvalho Aguiar, 500, 1°andar, sala 12. Bairro Cerqueira César. Zip code: 05403-000. São Paulo – SP, Brazil. Phone/fax: (11) 3085-1601. E-mail: [email protected]. The Brazilian Journal of Infectious Diseases 2008;12(5):358-361. © 2008 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

Studies analyzing semen by molecular biology techniques have reported difficulty in eliminating natural inhibitors present in the sample, which frequently leads to false-negative results [9]. Cassuto et al. [10], in a study of semen from 35 men, reported difficulty in removing inhibitors during HCV-PCR, with only five men testing positive, suggesting false-negative results. Various investigators have been able to detect HCV in semen, while others could not. The difficulty in isolating this virus from body fluids, especially semen, is probably due to the presence of inhibitors and the lack of standardized techniques and protocols for RNA extraction and reverse transcription and polymerase amplification (RT-PCR); these factors may contribute to false-negative results [9,11]. Our objective was to investigate whether HCV is present in semen samples from infected patients. Material and Methods Patient Selection Between June and December 2004, male patients with a clinical and laboratory diagnosis of HCV infection were recruited from the Hepatitis Outpatient Clinic of the Infectious Diseases Department. All recruited patients signed an informed consent agreement. Blood and Semen Collection and Preparation Blood was collected from patients into 10mL dry vacuum tubes, after an 8 to 12-h fast. Semen samples were obtained by self-masturbation, after a period of sexual abstinence of at least three days. Blood and semen samples were stored at 80°C until the time of use. In addition to an aliquot of total semen, fractions were isolated on 90 and 45% Percoll gradients. The samples were centrifuged for 30 min. at 3,000 rpm, and the following three phases were obtained: seminal plasma, leukocytes and spermatozoa.

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HCV in Semen

Serum All serum samples were analyzed with the qualitative Amplicor HCV test, version 2.0 (Roche Diagnostics Corp., Indianapolis, IN, USA). If positive, the blood samples were genotyped with INNO-LiPA HCV II kit (INNO-LIPA HCV, Versant Bayer, Tarrytown, NY, USA). Semen All semen samples were analyzed with the qualitative Amplicor HCV test, version 2.0 (Roche Diagnostics Corp., Indianapolis, IN, USA). After the end of the PCR-HCV reaction, the presence of HCV in semen samples was revealed by two methods: i) electrophoresis in agarose gel and ii) Roche Amplicor test by colorimetric determination. Electrophoresis Agarose Gel Detection The PCR-HCV products were detected by 2% Agarose Gel electrophoresis, stained with ethidium bromide and observed under ultraviolet light [13-15]. Amplicor HCV Test, v 2.0 The Amplicor HCV Test, v 2.0, is an RT-PCR in a manual, microwell format that amplifies a 244-nucleotide segment of the 5’-UTR of the HCV genome. The test was performed at all sites according to manufacturer’s instructions, as previously described. HCV RNA optical density (OD) values were interpreted as follows: 24 months

43 (57.3) 27 (36.0) 5 (6.7)

(54.7) (1.3) (10.7) (33.3)

*Occupational exposure to blood (needles, contaminated syringes) or sexual relationship with an infected individual. † RL – relapser; NR – non-responder. ‡ IFN – interferon; RBV – ribavirin; PEG – pegylated interferon.

Table 2. Post-treatment fibrosis qualitative evolution. N

%

N* P R Total

43 18 14 75

57.3 24.0 18.7 100.0

N

%

Non-regression* Regression Total

56 19 75

74.7 25.3 100.0

*Non-regression: patients with impairment or stabilization of inflammatory activity.

patients who had shown fibrosis regression and progression (p=0.024), with a higher regression rate and a lower progression rate of liver inflammation in the former compared to the latter. There was information available about previous alcohol consumption in the medical records of the 44 patients: 14 (31.8%) reported no alcohol use, 15 (34.1%) reported occasional use and 15 (34.1%) admitted daily use of alcoholic beverages. Statistical differences in alcohol consumption between mild-to-moderate (F0/F1/F2) and advanced (F3/F4) fibrosis patients were not found (p=0.660). Among the 15 patients who admitted daily use of alcohol, 13 (86.7%) showed mild-to-moderate liver fibrosis before treatment.

(40.0) (10.7) (9.3) (40.0)

Evolution

Evolution

*N – non-progression (stabilization) P – progression; R – regression.

Discussion The main obstacle against including patients was the unavailability, for various reasons, of liver biopsies for revision by the pathologist. This occurred in 355 cases. Some other reasons for not including patients were: patients that had been treated by other health facilities outside of the ERIDI; patients who had been under irregular treatment, with discontinuity or reduced doses of the treatment drugs; patients whose time interval between the first biopsy and treatment exceeded one year; and patients who had made the liver biopsy less than six months after the end of treatment. As a retrospective case series on a medical records revision basis, this study has some serious limitations. The most important of them comes from the lack of standardization of the medical record information. Another weakness originates from the adoption of a consensual and universal treatment that has prevented the use of a control group of patients without treatment for comparison. Lastly, the period of time during which the patients were under observation after treatment was significantly shorter than the pre-treatment period. This may overestimate the therapeutic benefits in cases of slowly progressing diseases. Statistical associations were found among treatment, hepatic fibrosis non-progression and decreases in the annual

Table 3. Post-treatment fibrosis qualitative evolution according to pre-treatment fibrosis stages. Pre-treatment fibrosis stages

Stages 0, 1 and 2 Stages 3 and 4 Total

Fibrosis evolution n (%)

P value

Non progression

Progression

Regression

32 (58.2) 11 (55.0) 43 (57.3)

17 (30.9) 1 (5.0) 18 (24.0)

6 (10.9) 8 (40.0) 14 (18.7)

* Pearson’s chi-square.

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0.005*

Histological Effect of Interferon

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365

Figure 1. Pre and post-treatment fibrosis stage. The shadow zone indicates stabilization of fibrosis. Above and to the right of the shadow zone indicates fibrosis progression. Below and to the left indicates fibrosis regression. Post- treatment fibrosis – n (%) Pretreatment fibrosis

0

1

0

3

2

1

(4.0)

(2.7)

(1.3)

3

14

4

1

1

(4.0)

(18.7)

(5.3)

(1.3)

(1.3)

3

15

6

2

(4.0)

(20.0)

(8.0)

(2.7)

1

3

8

1

(1.3)

(4.0)

(10.7)

(1.3)

4

3

(5.3)

(4.0)

1

2

3

2

4

3

4

Figure 2. Fibrosis progression before and after treatment. 80 70 No progression

60

Progression

50 40 30 20 10 0 Pre-treatment

Post-treatment

Table 5. Inflammatory activity evolution according to fibrosis evolution. Fibrosis evolution

Regression Stabilization Progression Total

Inflammatory activity evolution - N (%) Regression

Stabilization

7 (50)* 10 (23.3) 2 (11.2) 19

7 (50) 25 (58.1) 8 (44.4) 40

*p = 0.024 (Pearson’s chi-square).

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Progression 0 8 (18.6) 8 (44.4)* 16

Total 14 (100) 43 (100) 18 (100) 75

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Histological Effect of Interferon

fibrosis progression rate, in comparison with the chronic hepatitis C natural history. Similar results have been found in other studies [8,18,20,30]. The observation of hepatic fibrosis regression in 14 patients is a very important piece of information, since spontaneous regression of hepatic fibrosis is not usually seen in patients without treatment. We speculate that the regression in fibrosis is due to an antifibrogenic effect of interferon, regardless of its antiviral action. Poynard et al. demonstrated that hepatitis C is indeed a progressive fibrotic disease, which is the reason for its morbidity and mortality [4]. Therefore, a therapeutic intervention to decrease or revert hepatic fibrosis progression will probably have a relevant clinical impact. In our study, seven out of 14 patients with fibrosis regression also showed posttreatment inflammatory activity regression, while the other seven patients remained at the same activity level. It seems reasonable to attribute fibrosis regression in the latter group to an intrinsic antifibrogenic effect. On the other hand, the relevant fibrosis stabilization rates in patients with mild-to-moderate, as well as advanced fibrosis (58.2% and 55.0%, respectively), are probably due to the anti-inflammatory action of ribavirin and interferon. As a matter of fact, among the 75 patients, only 21 (28%) had made use of standard interferon (IFN) alone, while the other 54 (72%), had made use of ribavirin along with IFN or PEG, with the possibility that the ribavirin indirectly facilitated the interferon antifibrogenic effect through its antiinflammatory action. It has been noticed among patients with advanced fibrosis before treatment, that there is a higher proportion of patients with fibrosis regression and a larger reduction in the annual fibrosis progression rate, after treatment in comparison with patients who had mild-to-moderate fibrosis. Again, there was a good correlation between the qualitative evolution and the annual fibrosis progression rate. Poynard et al. found similar results among 1,509 French patients, though they used a different methodology for defining mild-to-moderate and advanced fibrosis [20]. However, in our study, the small number of patients with advanced fibrosis did not make it possible to make conclusions from the results. Treatment effects on inflammatory activity evolution were less evident. About 75% of the patients worsened or did not present any variation in hepatic inflammatory activity, in disagreement with several other studies that reported on the anti-inflammatory effect of chronic hepatitis C treatment [8,17,31-35]. Even so, 66% of the patients showed null or mild degrees of inflammatory activity after treatment, foreseeing an absence of fibrosis progression on the short and probably the mid-term. Studies of Poynard et al. about the natural history of hepatitis C show that the degree of inflammatory activity is less associated with the time of disease than fibrosis, which reflects the oscillating inflammatory activity due to infection [4]. For a better measurement of the treatment’s impact upon inflammatory

BJID 2008; 12 (October)

liver activity, a post-treatment hepatic biopsy should have been made, preferably right away after the end of the treatment, or within the first six months, as has been done in other studies [17,32-34]. Finally, we did not find histological benefits for longer exposure to interferon in terms of fibrosis or inflammatory activity improvements. A possible reason was the small number of patients exposed to interferon over 24 months. In conclusion, the significant proportion of patients who have not shown fibrosis progression, associated with the significant fibrosis progression rate reduction after an interferon-based treatment, support the hypothesis of an antifibrogenic effect of interferon, associated or not with ribavirin, in patients that still carry the virus after treatment. We conclude that long-term reduced dosages of interferon for patients with advanced fibrosis and who have no virological response to classic treatment would help prevent liver damage. Acknowledgements The authors thank the Department of Pathology for logistical support and the Documentation and Medical Records Service of Emilio Ribas Infectious Diseases Institute for allowing the revision of liver biopsies and medical charts of the patients. References 1. WHO Consultation. Global surveillance and control of hepatitis C: report of a WHO consultation organized in collaboration with the Viral Hepatitis Prevention Board, Antwerp, Belgium. J Viral Hepat 1999;6:35-47. 2. Focaccia R., Conceição O.J., Sette Jr. H., et al. Estimated prevalence of viral hepatitis in the general population of the municipality of São Paulo, measured by a serologic survey of a stratified, randomized and residence-based population. Braz J Infect Dis 1998;2(6):269-84. 3. Centers for Disease Control and Prevention. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. MMWR 1998;47(RR-19):1-40. 4. Poynard T., Bedossa P., Opolon P. for the OBSVIRC, and METAVIR, CLINIVIR, and DOSVIRC groups. Natural history of liver fibrosis progression in patients with chronic hepatitis C. Lancet 1997;349:825-32. 5. Seeff L.B. Natural history of chronic hepatitis C. Hepatology 2002;36(Suppl.1):S35-46. 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 randomized trial. Lancet 2001;358:958-65. 7. Fried M.W., Shiffman M.L., Reddy R., et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347:975-82. 8. Poynard T., McHutchison 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. 9. EASL. Consensus Statement. EASL International Consensus Conference on Hepatitis C. J Hepatol 1999;30:956-61. 10. National Institutes of Health Consensus Development Conference Panel Statement: Management of Hepatitis C. Hepatology. 1997;26:2S-10S.

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11. Wejstal R., Alaeus A., Fischler B., et al. Chronic hepatitis C: updated Swedish consensus. Scand J Infect Dis 2003;35(8):445-51. 12. Li D., Friedman S.L. Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. J Gastroenterol Hepatol 1999;14(7):618-33. 13. Sakaida I., Nagatomi A., Hironaka K., et al. Quantitative analysis of liver fibrosis and stellate cell changes in patients with chronic hepatitis C after interferon therapy. Am J Gastroenterol 1999;94(2):489-96. 14. Duchatelle V., Marcellin P., Giostra E., et al. Changes in liver fibrosis at the end of alpha interferon therapy and 6 to 18 months later in patients with chronic hepatitis C: quantitative assessment by a morphometric method. J Hepatol 1998;29:20-8. 15. Abe S., Tabaru A., Ono M., et al. High-dose interferon-a therapy lowers the levels of serum fibrogenesis markers over 5 years in chronic hepatitis C. Hepatology Research 2003;25:22-31. 16. Caballero T., Pérez-Milena A., Masseroli M., et al. Liver fibrosis assessment with semiquantitative indexes and image analysis quantification in sustained-responder and non-responder interferon-treated patients with chronic hepatitis C. J Hepatol 2001;34:740-7. 17. Shiffman M.L., Hofmann C.M., Contos M.J., et al. A randomized, controlled trial of maintenance interferon therapy for patients with chronic hepatitis C virus and persistent viremia. Gastroenterology 1999;117:1164-72. 18. Sobesky R., Mathurin P., Charlotte F., et al. Modeling the impact of interferon alfa treatment on liver fibrosis progression in chronic hepatitis C: a dynamic view. Gastroenterology 1999;116:378-86. 19. Guerret S., Desmoulière A., Chossegros P., et al. Long-term administration of interferon-[alpha] in non-responder patients with chronic hepatitis C: follow-up of liver fibrosis over 5 years. J Viral Hepat 1999;6(2):125-33. 20. Poynard T., McHutchison J.G., Davis G.L., et al. Impact of interferon alfa-2b and ribavirin on progression of liver fibrosis in patients with chronic hepatitis C. Hepatology 2000;32:1131-7. 21. Kojima H., Hongo Y., Harada H., et al. Long-term histological prognosis and serum fibrosis markers in chronic hepatitis C patients treated with interferon. J Gastroenterol Hepatol 2001;16:1015-21. 22. Pol S., Carnot F., Nalpas B., et al. Reversibility of hepatitis C virus-related cirrhosis. Human Pathol 2004;35(1):10712.

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23. Poynard T., Marcellin P., Lee S.S., et al for the International Hepatitis Interventional Therapy Group (IHIT). Randomised trial of interferon alfa-2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alfa-2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 1998;352:1426-32. 24. McHutchison J.G., Gordon S.C., Schiff E.R., et al. For the Hepatitis Interventional Therapy Group. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998;339:1485-92. 25. Alter M.J., Kruszon-Moran D., Nainan O.V., et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999;341:556-62. 26. Ferrell G.C., Bacon B.R., Goldin R.D. Lymphoblastoid interferon alfa-n1 improves the long-term response to a 6-month course of treatment in chronic hepatitis C compared with recombinant interferon alfa-2b: results of an international randomized controlled trial. Hepatology 1998;27:1121-7. 27. Gayotto LCC e Comitê SBP/SBH. Visão histórica e consenso nacional sobre a classificação das hepatites crônicas. GED. 2000;19(3):137-40. 28. Snedecor GW & Cochran WG. Statistical Methods. Sixth Edition, 1967. The Iowa State University Press. 29. StataCorp. Stata statistical Software. Release 7.0. College station, TX: Stata Corporation, 2001. 30. Shiratori Y., Imazeki F., Moriyama M., et al. Histologic improvement of fibrosis in patients with hepatitis C who have sustained response to interferon therapy. Ann Intern Med 2000;132:517-24. 31. The French METAVIR Cooperative Study Group. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology 1994;20:15-20. 32. Bedossa P., Poynard T. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology 1996;24:289-93. 33. Poynard T., Bedossa P., Chevallier M., et al. A comparison of three interferonalfa-2b regimens for the long-term treatment of chronic non-A, non-B hepatitis. N Engl J Med 1995;332:1457-62. 34. Shiffman M.L., Hofmann C.M., Thompson E.B., et al. Relationship between biochemical, virological, and histological response during interferon treatment of chronic hepatitis C. Hepatology 1997;26:780-5. 35. Marcellin P., Boyer N., Gervais A., et al. Long-term histologic improvement and loss of detectable intrahepatic HCV RNA in patients with chronic hepatitis C and sustained response to interferon-α therapy. Ann Intern Med 1997;127:875-81.

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Cost-Effectiveness of Entecavir versus Lamivudine for the Suppression of Viral Replication in Chronic Hepatitis B Patients in Brazil Anna Maria N. Costa1, Gilbert L.’Italien2, Marcelo Eidi Nita1 and Evaldo Stanislau A. Araujo 3 Global Development and Medical Affairs da Bristol-Myers Squibb Company, São Paulo; 2Global Epidemiology and Outcome Research, Bristol-Myers Squibb, Wallingford, CT, USA; 3Infectious Diseases Division of Clinical Hospital of Medical School of São paulo University; São Paulo, SP, Brazil 1

Hepatitis B virus infection is an important public-health issue. Chronic patients have a higher risk of death due to complications, which increases health-care expenses in. Cost-effectiveness analysis of entecavir (ETV) versus lamivudine (LVD) for treatment of chronic hepatitis B, in e antigen (AgHBe)-positive and negative patients, based on two phase 3, controlled and randomized studies. A decision analysis model was developed, using the following endpoints: cost per patient with undetectable viral load and cost per quality life year (QALY) gained. Risks for complications (compensated or decompensated cirrhosis and hepatocellular carcinoma) were based on the cohort study REVEAL, published in 2006. The REVEAL parameters were applied to the results of the viral load levels obtained from the clinical assay data. The complication costs were based on a study of the disease cost conducted in Brazil, in 2005. The cost data were obtained predominantly from Sistema Único de Saúde [SUS - Brazilian public health system] payment tables and drug price lists. The utility data were obtained from literature and life expectancy information was based on IBGE data. The analysis perspective was that of SUS. A discount rate of 3% per year was used. For the horizon of time of 10 years, the ETV had an incremental cost of approximately two million Brazilian Reais (R$) compared to LVD. Reducing the number of complications, ETV treatment reduced costs by around 3 million, reducing final costs by 1 million, for AgHBe-positive patients. ETV also reduced the incremental cost per QALY gained. ETV was found to be the most cost-effective alternative for AgHBe-positive and negative patients. Key-Words: Chronic hepatitis B, cost, treatment.

Hepatitis B virus (HBV) infection is an important public health issue, due to its potential to evolve to chronic forms of the disease (CHB), characterized by persistence of the hepatitis B virus surface antigen (AgHBs); the chronic form of the disease has important economic impacts for society. In addition to AgHBs, other antigens are found in the blood, such as the e antigen (AgHBe). Independent of whether infections are positive for this antigen, hepatic disease continues to progress, affecting approximately 350 million people worldwide. In Brazil, at least 15% of the population have already been in contact with hepatitis B virus and 1% present chronic disease. Subjects with chronic infection have a higher risk of death for disease complications, such as hepatic cirrhosis and hepatocellular carcinoma (HCC), which also contribute to increased costs due to morbidity [1-3]. CHB treatment has as a primary objective prevention of cirrhosis or HCC. According to the latest U.S. algorithm for the treatment of this disease, prevention can be attained through viral DNA suppression to minimal values during the longest time possible [4] . At the moment, there are two drugs available for CHB treatment in the Programa Nacional de Hepatites Virais [National Program of Viral Hepatitis], of the Ministry of Health: interferon-alpha and lamivudine (LVD). LVD has drawbacks, including a high rate of occurrence of Received on 11 April 2008; revised 25 September 2008. Address for correspondence: Dr. Anna Maria N. Costa, MD, PhD. Email: [email protected]. Telephone : 11 3882 2374. BristolMyers Squib Farmacêuticas S/A. Rua Carlos Gomes, 924, Santo Amaro – SP. Zip code:: 04743-903. The Brazilian Journal of Infectious Diseases 2008;12(5):368-373. © 2008 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

mutant virus, which has been generating discussions and preoccupations by medical societies. The incidence of CHB complications and the limitation of therapies available for the treatment of the CHB disease that reduce and control the viral load are factors contributing to increases inhealth expenses in the Sistema Único de Saúde (SUS), due to CHB. Entecavir (ETV) is the newest oral antiviral agent approved for the treatment of this disease. It is a carbocyclic analogue of 2’-deoxyguanosine, with accentuated in vitro and in vivo activity against viral DNA polymerase, inhibiting viral replication [5]. It has been found to have greater efficacy than LVD, with fewer long-term complications. In several studies, ETV presented favorable profiles of efficacy, safety and resistance [6-8]. In a recent metanalysis [6], which evaluated AgHBe-positive and negative patients, an efficacy comparison was made for ETV, LVD and adefovir, after 48-52 weeks of treatment. In HBeAg-positive patients, ETV more effectively reduced viral DNA levels than did LVD or adefovir (p